Identifying widespread and recurrent variants of genetic parts to improve annotation of engineered DNA sequences

Engineered plasmids have been workhorses of recombinant DNA technology for nearly half a century. Plasmids are used to clone DNA sequences encoding new genetic parts and to reprogram cells by combining these parts in new ways. Historically, many genetic parts on plasmids were copied and reused without routinely checking their DNA sequences. With the widespread use of high-throughput DNA sequencing technologies, we now know that plasmids often contain variants of common genetic parts that differ slightly from their canonical sequences. Because the exact provenance of a genetic part on a particular plasmid is usually unknown, it is difficult to determine whether these differences arose due to mutations during plasmid construction and propagation or due to intentional editing by researchers. In either case, it is important to understand how the sequence changes alter the properties of the genetic part. We analyzed the sequences of over 50,000 engineered plasmids using depositor metadata and a metric inspired by the natural language processing field. We detected 217 uncatalogued genetic part variants that were especially widespread or were likely the result of convergent evolution or engineering. Several of these uncatalogued variants are known mutants of plasmid origins of replication or antibiotic resistance genes that are missing from current annotation databases. However, most are uncharacterized, and 3/5 of the plasmids we analyzed contained at least one of the uncatalogued variants. Our results include a list of genetic parts to prioritize for refining engineered plasmid annotation pipelines, highlight widespread variants of parts that warrant further investigation to see whether they have altered characteristics, and suggest cases where unintentional evolution of plasmid parts may be affecting the reliability and reproducibility of science. Author Summary Plasmids are used in molecular biology and biotechnology for a wide variety of tasks such as cloning DNA, expressing recombinant proteins, and creating vaccines. One challenge in working with plasmids is that there has been a long, and often lost history of pieces of plasmids being copied and remixed by researchers to create new plasmids. Current databases used for annotating key genetic parts in plasmids are incomplete, especially with respect to cataloguing closely related versions of parts that can have very different characteristics. Some genetic part variants have arisen due to purposeful editing while others are the result of unplanned mutations and evolution. When a researcher finds differences between a database sequence and a genetic part in their newly constructed plasmid, it is often unclear how and when it arose and whether it will affect their experiments. We identified 217 genetic part variants that are either widespread or have likely arisen independently more than once on plasmids due to convergent evolution or engineering. We propose that these variants should be prioritized for inclusion in curated databases of engineered DNA sequences and for functional characterization to improve the reliability and reproducibility of science.