Expansion and Optimization of DIVA DNA Sequence Validation Services

Advances in DNA sequence validation technology has enabled the Joint BioEnergy Institute (JBEI) to offer high-throughput plasmid sequencing to its researchers. However, the types of compatible samples have not significantly expanded. This lack of compatibility leads to time and materials wasted on purifying or transforming a plasmid into E. coli. Here, we sought to update the current sequencing protocol to include the most utilized species and plasmid types, including E. coli cultures with medium and low copy plasmids, A. tumefaciens, P. putida, S. cerevisiae, and P. pastoris. Based on previous runs, the laboratory determined that a baseline coverage greater than 60x over the whole plasmid yielded the most accurate reads. This project showed that E. coli was the most robust species, with sequencing succeeding for all liquid cultures, regardless of copy number, and for resuspended colonies picked directly from agar plates. The remaining species experienced varying levels of coverage failure. However, minimal modification to the existing sequencing workflow, such as longer lysing times or the use of higher copy plasmids, could increase overall coverage. Overall, the sequence validation services provided by JBEI have numerous advantages over traditional Sanger sequencing with third-party companies. By expanding the compatible sample types, we are making this efficient, cost-effective sequencing service more accessible to JBEI’s researcher community.