Southern‐by‐Sequencing: A Robust Screening Approach for Molecular Characterization of Genetically Modified Crops

Abstract

Molecular characterization of events is an integral part of the advancement process during genetically modified (GM) crop product development. Assessment of these events is traditionally accomplished by polymerase chain reaction (PCR) and Southern blot analyses. Southern blot analysis can be time-consuming and comparatively expensive and does not provide sequence-level detail. We have developed a sequence-based application, Southernby-Sequencing (SbS), utilizing sequence capture coupled with nextgeneration sequencing (NGS) technology to replace Southern blot analysis for event selection in a high-throughput molecular characterization environment. SbS is accomplished by hybridizing indexed and pooled whole-genome DNA libraries from GM plants to biotinylated probes designed to target the sequence of transformation plasmids used to generate events within the pool. This sequence capture process enriches the sequence data obtained for targeted regions of interest (transformation plasmid DNA). Taking advantage of the DNA adjacent to the targeted bases (referred to as nextto-target sequence) that accompanies the targeted transformation plasmid sequence, the data analysis detects plasmid-to-genome and plasmid-to-plasmid junctions introduced during insertion into the plant genome. Analysis of these junction sequences provides sequence-level information as to the following: the number of insertion loci including detection of unlinked, independently segregating, small DNA fragments; copy number; rearrangements, truncations, or deletions of the intended insertion DNA; and the presence of transformation plasmid backbone sequences. This molecular evidence from SbS analysis is used to characterize and select GM plants meeting optimal molecular characterization criteria. SbS technology has proven to be a robust event screening tool for use in a highthroughput molecular characterization environment.

High efficiency Agrobacterium‐mediated site‐specific gene integration in maize utilizing the FLP‐FRT recombination system

“…Over 100 T0 RMCE events, were subjected to SbS analysis (Zastrow‐Hayes et al ., ). Only events with fully intact donor DNA insertions (flanked by intact FRT 1 / 87 sites) and no unintended DNA sequence were considered SbS‐pass events.…”

“…This design is similar to one described for soybean SSI (Li et al, 2009(Li et al, , 2010. The RTLs were molecularly characterized using Southern-by-Sequencing TM technology, hereafter referred to as SbS analysis (Zastrow-Hayes et al, 2015). SbS utilizes capture-based target enrichment of samples prior to next-generation sequencing (NGS) and is used to determine the insertion sequence and intactness of the inserted DNA at the target site.…”

“…The details of the primer probe used for molecular analysis to identify the intended RMCE are described in Table S5a. The transformants identified with the intended donor DNA insertions (RMCE) were subjected to Southern-by-Sequencing as previously described (Zastrow-Hayes et al, 2015). Events with fully intact donor DNA flanked by the heterologous FRT pairs absent for unintended DNA sequence were identified as SbS pass events.…”

Foreign DNA detection by high-throughput sequencing to regulate genome-edited agricultural products

“…Among high-throughput sequencing technologies, SBS has long been used for molecular biology for more than a decade. It is relatively mature enough for routine work and several investigations for practical applications of this method have been carried out for GMOs [11][12][13][14][15][16] . In this study, we showed that in addition to past conventional methods, SBS is also immediately applicable to GEAP regulation.…”

Next generation DNA sequencing based strategies; towards a new era for the traceability of endangered species and genetically modified organisms

“…In a recent study, using this approach, three 70 nucleotides long biotinylated probes were designed to target the extremities of T-DNA sequences to rapidly locate the T-DNA insertion sites in 55 out of 64 mutant Arabidopsis plants [259]. A similar approach, called Southern-by-Sequencing (SbS), was used to characterise the insertion sites in GM crops [260]. In both the approaches, information of the construct close to the insertion sites is necessary for characterisation, which is typically not the case in unknown GMO identification.…”