I've been learning a lot about molecular biology recently, and am about to run some procedures related to plasmid isolation, gene cloning, and expression. Of note, the resources I've read, from AddGene, and The Bumbling Biochemist (excellent Youtube channel) emphasize sending your plasmids in for sequencing once complete, due to all the errors that can occur.
I am wondering if the plasmids the researchers here tested simply weren't sequenced. This wouldn't explain all classes of errors (like toxic protein production), but it may explain some. When you order from AddGene, they provide both the depositor's sequence results, if available, and their own results. The paper doesn't mention AddGene specifically; I'm curious how their plasmids compare to the ones tested.
Regarding toxic proteins: It seems like that would be a straightforward software addition to pass sequencing results through.
Before these companies like plasmidsaurus that do whole-plasmid sequencing for relatively cheap with nanopore, people generally only sequenced a region of interest using sanger sequencing. The rest of the plasmid was assumed to be mostly correct, as long as it grows on a bacterial resistance. As noted in the article, the rise of nanopore-based whole-plasmid sequencing has reduced a lot of these types of errors.
I figure the vast majority of plasmids are used for mundane stuff like GFP expression. Not really worth sending in for an analysis because everyone just redoes the experiment if it fails to express anyway.
I am wondering if the plasmids the researchers here tested simply weren't sequenced. This wouldn't explain all classes of errors (like toxic protein production), but it may explain some. When you order from AddGene, they provide both the depositor's sequence results, if available, and their own results. The paper doesn't mention AddGene specifically; I'm curious how their plasmids compare to the ones tested.
Regarding toxic proteins: It seems like that would be a straightforward software addition to pass sequencing results through.