Comparative evaluation of gene copy number estimation techniques in genetically modified crops: insights from Southern blotting, qPCR, dPCR and NGS

Gene copy number is crucial for understanding genomic architecture and its implications in plant and animal genetics (Alonge et al., 2020; Castagnone-Sereno et al., 2019). In agriculture, variations in gene copy number (CNVs) are vital as they affect yield, stress resistance and metabolic capabilities (Yuan et al., 2021). Transgenesis, involving the introduction of foreign DNA into plant genomes, has revolutionized agriculture by creating genetically modified (GM) plants with desirable traits. Assessing gene copy numbers in GMOs ensures stability and expression of introduced traits and is crucial for regulatory compliance and biosafety assessments (Liang et al., 2022). Evaluating gene copy numbers in transgenic plants is technically challenging due to variability in transgene integration events (Faure, 2021). Various techniques like Southern blotting (SB), quantitative real-time PCR (qPCR), digital PCR (dPCR) and paired-end whole-genome sequencing (PE-WGS) have been reported for gene copy number determination (Cusenza et al., 2021). However, no systematic comparison of these four methods has been reported, especially concerning PE-WGS.

Here, we performed a comparative benchmarking of gene copy number assessment techniques, including SB, qPCR, dPCR and PE-WGS, employing 4 GM crop events (FG72 soybean, 12-5 maize, G6H1 and G281 rice) as examples (Figure 1a; Data S1).

Figure 1

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(a) The workflow of the benchmarking study of gene copy number estimation, the diagrams of exogenous gene cassettes of the tested GM events (FG72, G281, G6H1 and 12-5), and the position of hybrid probes in Southern blotting analysis. (b) Southern blotting analysis of four events with various restriction enzymes. GM, GM event; M, DNA marker; P, positive control; WT, the corresponding recipient line of GM event. (c) The constructed standard curves of qPCR assays employing corresponding plasmid calibrators of exogenous and endogenous genes. (d) Summarizes the copy numbers of transgenes determined from Southern blotting, qPCR, ddPCR and PE-WGS analysis. (e) Advantages and disadvantages of the four methods in transgene copy number evaluation. dPCR, digital PCR; GM, genetically modified; PCR, polymerase chain reaction; PE-WGS, paired-end whole-genome sequencing; qPCR, quantitative PCR.

In SB analysis, we used various restriction endonucleases for genomic DNA digestion. For the event G6H1, BamHI, SacI, KpnI and StuI revealed one copy of cry1Ab/vip3H and G6epsps. The G281 event showed a single copy of hLF but uncertain G6epsps copy numbers (one or two). FG72 displayed inconsistent band patterns for 2mepsps and hppdPfW336, suggesting copy numbers of one or two. Maize 12-5, analysed with KpnI and XbaI, indicated a single copy of G10epsps and cry1Ab/cry2Aj (Figure 1b, Table S1).

We employed absolute quantification via qPCR, using endogenous reference genes SPS, Lectin and zSSIIb for rice, soybean and maize, respectively. All assays were validated for high efficiency and precision (Figure 1c, Table S2). Quantitative real-time PCR results showed G6H1's G6epsps and cry1Ab/vip3H had values of 0.98 and 0.96; G281's G6epsps and hLF were 1.68 and 1.54; FG72's 2mepsps and hppdPfW336 were 1.72 and 1.67; maize 12-5's G10epsps and cry1Ab/cry2Aj were 0.81 and 0.83 (Table S3). These values suggest a single T-DNA fragment integration in G6H1 and 12-5 and two fragments in G281 and FG72.

Digital PCR provides absolute quantification by comparing target DNA to a reference gene. Results showed G6H1 had 0.94 and 0.97 copies for G6epsps and cry1Ab/vip3H. G281's G6epsps and hLF were 1.85 and 1.93. FG72's 2mepsps and hppdPfW336 were 1.69 and 1.68. 12-5's G10epsps and cry1Ab/cry2A were 0.57 and 0.59 (Table S4). These indicate single exogenous gene copies in G6H1 and 12-5 and dual copies in G281 and FG72.

In PE-WGS analysis, the sequencing depths for G6H1, G281, FG72 and 12-5 were 28.81, 28.91, 48.70 and 23.94, respectively (Table S5). Read counts for target genes were used to calculate copy numbers: G6H1 had 1.08 copies of G6epsps and 0.83 copies of cry1Ab/vip3H; G281 had 2.01 copies of G6epsps and 1.91 copies of hLF; FG72 had 1.80 copies of 2mepsps and 2.00 copies of hppdPfW336; 12-5 had 0.58 copies of G10epsps and 0.61 copies of cry1Ab/cry2Aj (Table S6).

Our systematic measurements of four GM events showed that all four techniques are suitable for this purpose to varying degrees. All methods accurately quantified single-copy genes; however, discrepancies emerged for multi-copy genes (Figure 1d). The strengths and limitations of each method concerning various aspects were summarized in Figure 1e.

Southern blotting is less accurate and sensitive for multi-copy genes. Southern blotting often underestimates multi-copy genes due to complex arrangements like tandem repeats and can overestimate due to incomplete digestion and cross-hybridization. qPCR, while more accurate than SB, struggles with high-copy genes due to resolution limits (around two-fold variation). Proper primer design and reaction optimization can still yield relatively accurate results. Digital PCR excels with high accuracy for multi-copy genes due to its partitioning capability, allowing it to detect minor changes in copy number, such as a 1.2-fold change from 5 to 6 copies (Whale et al., 2012). Paired-end whole-genome sequencing also provides precise quantification for multi-copy genes through adequate coverage and sophisticated data analysis tools, especially useful for genes with complex genomic rearrangements (Hehir-Kwa et al., 2018). Furthermore, PE-WGS has demonstrated high performance in elucidating the comprehensive molecular characterization of transgenic plants and animals. This includes identifying transgene insertion sites, flanking sequences, entire T-DNA integration structures and plasmid backbone presence, among other features.

Digital PCR and PE-WGS are most effective for distinguishing heterozygotes from homozygotes. Digital PCR offers absolute quantification without needing a standard curve, providing precise measurements. Paired-end whole-genome sequencing, through high-resolution mapping of paired-end reads, can differentiate between heterozygotes and homozygotes by analysing read depth. It is challenging to distinguish between homozygotes and heterozygotes with SB due to similar patterns from sequence homology. Quantitative PCR can distinguish heterozygotes from homozygotes based on Ct values, but it requires careful calibration and control and is influenced by PCR efficiency.

Southern blotting and PE-WGS require substantial amounts of DNA, whereas PCR-based methods need significantly less. Quantitative PCR necessitates high-quality DNA free from degradation and PCR inhibitors. Digital PCR is more tolerant of DNA degradation and inhibitors, delivering accurate quantification even with crude DNA extracts (Whale et al., 2012).

Quantitative PCR and dPCR are generally easier to set up and perform than SB and PE-WGS. SB is labor-intensive, involving several complex steps, including DNA digestion, transformation, hybridization and autoradiography. Paired-end whole-genome sequencing requires strict protocols, thorough DNA extraction, library preparation, machine sequencing and data analysis, necessitating skilled personnel in molecular biology and bioinformatics. Quantitative PCR involves optimizing conditions, constructing positive plasmid DNA, creating standard curves and testing, requiring normal molecular laboratory skills. Digital PCR entails sample partitioning using instruments that streamline the process into one platform.

Quantitative PCR requires the least technical expertise, primarily understanding primer design and qPCR setup. Digital PCR needs moderate expertise, especially for sample emulsification or droplet/well creation. Southern blotting demands high technical skills, particularly for DNA digestion, gel electrophoresis, transfer and hybridization. Paired-end whole-genome sequencing requires significant expertise in data analytics and bioinformatics, besides library preparation and sequencing skills. The experimental duration of qPCR and dPCR is faster than SB and PE-WGS, typically concluding within a day. Southern blotting and PE-WGS require at least 3 days, respectively. Digital PCR is quicker than qPCR because it does not require a standard curve.

Costwise, SB is relatively cheap owing to lower reagent costs and basic equipment needs. Quantitative PCR has a medium cost, with moderately expensive reagents and higher throughput, which reduces per-sample costs. Digital PCR is more costly due to expensive equipment and lower throughput but provides absolute quantification without standard curves. Paired-end whole-genome sequencing is the most expensive, justified by its comprehensive genomic characterization capabilities beyond single gene copy estimation.

We propose prioritizing dPCR and PE-WGS for precise gene copy number analysis. Paired-end whole-genome sequencing is especially suited for assessing multiple gene copies within a sample, while dPCR is optimal for smaller quantities per sample, offering robust tools for genomic research and biotechnological applications.