Sanger sequencing is widely used due to its reliability and accuracy. However, it presents key limitations when applied to high-throughput or full-length variant analysis:

• Limited read length (~800–1000 base pairs), which may not capture entire constructs

• Low scalability, as each variant must be sequenced individually

• Manual processing, including read alignment and mutation mapping

• Increased time and cost with growing library size

• Not ideal for pooled or mixed-sample sequencing

These factors make Sanger sequencing inefficient for modern directed evolution workflows, especially when analyzing large mutant libraries.

Long-read platforms such as Oxford Nanopore address many of the limitations inherent to Sanger sequencing by enabling high-throughput, full-length analysis:

• Extended read lengths, capable of covering entire plasmids or gene constructs

• High-throughput potential, suitable for complex or pooled libraries

• Single-molecule resolution, allowing detection of rare variants

• Fewer preprocessing steps, with reduced need for tiling or assembly

• Plasmidsaurus Premium PCR offers amplification-free sequencing and delivers raw .fastq files directly for flexible downstream use

This approach is ideally suited for directed evolution experiments that require full-length, high-resolution variant identification.

Remaining Challenges in Data Analysis Despite improvements in sequencing technology, data analysis remains a barrier — particularly for research groups without dedicated computational expertise. Commercial platforms like Geneious offer graphical interfaces for demultiplexing and consensus generation, but: 

• Incur substantial licensing costs (e.g., ~$200/year/student) 

• Require manual processing of individual samples 

• Are not optimized for high-throughput variant analysis