Continuing with the LISA workshop, tonight I watched another session. This one was by a postdoc, Andrew Wilson, at Pacific Northwest National Laboratory. They have been working on plasmid and microbe sequencing. The title of the session was “Leveraging Long Reads for Synthetic Biology.” Wilson and team are using Gibson assembly and synthetic biology techniques. They have used services to sequence their constructs and isolates. Sequencing with Plasmidsaurus helps them confirm plasmids for not much more than “a few Sanger runs.” Wilson spoke about the benefits of plasmid sequencing is “when things go wrong.” In one example, Wilson shared that they were using an antibiotic that was stronger and most of the plasmids from one batch were duplicated in the backbone! In another case, they identified that an elution buffer stock had nucleases because there were many reads that were smaller than the plasmid expected size. Plasmidsaurus is also used by Wilson to sequence isolates. In one example, Wilson sequenced an isolate that grows on meta tyrosine. Plasmidsaurus sequenced the genome and helped assemble a single contig with very low contamination. Wilson spoke about the REGAiN – Methanotroph Engineering for Lanthamide Biomining. Wilson noted that regular ways of getting lanthanide from soil are expensive. Lanthanides are thought to not be very abundant in nature. However, microorganisms that use them have been identified. To engineer wild microbes in order to put DNA into these organisms, researchers include/engineer landing sites. Methylation patterns and restriction modification systems can be hard to predict and limit introduction of DNA into these organisms of interest. Wilson described PacBio and Nanopore sequencing as options for detecting modifications. Wilson and team used gDNA reads and whole genome amplification (isothermal) to compare methylation. They used the difference in pico amps in signal to distinguish strand-specific differences. The team identified a restriction system and was able to delete that gene to make a more genetically tractable organism. Next, Wilson spoke about poolGLAss – a system for optimizing expression of pathways with pooled genomic libraries. Pools of genomic libraries are used to identify antibiotic systems. This system has been adapted to PacBio systems for circular consensus sequencing of colonies. They can now use a liquid handler to print 384 colonies on plates and try to identify desired phenotypes. Wilson noted that now they have too much to analyze! One question after the session was whether they had experienced issues with sequencing with Plasmidsaurus. Wilson has sequenced only a handful of genomes without any issues. Another concern was that even with high coverage, long-read only assemblies may have errors. The last question was about detection of modifications and how this can be done with Dorado more efficiently now. I am learning by watching these sessions and hope to use this knowledge for our genomes for BIT 295.
