Table 2 Comparison of the key features of Southern blots and NGS

Key characteristics

Experimental design

• Event specific

• Standard

Procedure

• Labor-intensive

• High throughput

Reproducibility

• High but multiple manual procedures may cause variation in the results

• High

Data analysis tools

• Manual

• Automated software tools for data analysis

Data output

• Blot images

• DNA fragment level of details

• Mapping/visualization of sequence reads

• Nucleotide level of details

Accuracy

• High in combination with Sanger sequence

• High

Throughput

• Low

• High

Molecular characterization

Presence/absence of T-DNA

• Confirmation through genome digestion with restriction enzymes and analysis with T-DNA probe, validated by band presence

• Confirming mapping regions by aligning the entire nucleotide sequence reads to the plasmid backbone

Insertion site

• Determination through PCR primer walking using T-DNA primers

• Confirmation by mapping plasmid flanking region reads to the genome reference

Sequence structure

• Separate analysis of nucleotide sequences within the T-DNA region

• Confirmation of sequence structure by mapping genomic position and T-DNA reads to the plasmid

Replication number

• Determination via count of Southern bands

• Verification through reference mapping using reads from plasmid backbone flanking regions

Unintended inserted DNA

• Inability to confirm using only T-DNA as a probe

• Probing of the entire plasmid for confirmation

• Detection possible during mapping to the plasmid backbone

Endpoint verification

Copy number and integrity

• Confirming copy number based on the pattern of bands detected using specific probes and the size of fragments

• Verifying copy number and integrity of insertions by examining the number of reads and junctions matching the T-DNA region indicated on the map

Absence of backbone

• Confirming the absence of backbone insertion by detecting bands using specific probes at the respective positions

• Verifying the absence of backbone insertion by observing the absence of read sequences in the backbone region of the same plasmid map where T-DNA has been confirmed

Stability across generations

• Determining stability by confirming if bands observed in characterized generations are consistently observed at the same positions across multiple generations using specific probes

• Confirming stability by examining consistency in T-DNA read mapping, number of junctions, and the absence of backbone across characterized and multiple generations