Last year was an exciting one for Addgene as we introduced our long-awaited viral service, but we haven’t forgotten about our plasmids! Now, we’re improving our quality control processes using next-generation sequencing (NGS) services provided by seqWell. This new QC process will bring you full sequence data for new plasmids entering the repository. Read on to learn more about how this process works and what you can expect to see on our plasmid pages.
Our tried-and-true method for quality control uses Sanger sequencing, described in a previous blog post. This process requires a primer to anneal upstream of the region of interest, and generally delivers 500-700 bases of useful sequence. We customarily used 1-2 Sanger reactions to verify features of interest, adding extra reactions to verify other features. Although Sanger sequencing is fast and inexpensive, it doesn’t give us a full picture of a plasmid.
Through our collaboration with seqWell, Addgene can now obtain full plasmid sequences using NGS. This extra information makes it easier to understand what features, restriction sites, etc. a plasmid contains. Briefly, NGS technologies create many short, ~150-500 bp "reads" across an entire plasmid, and these reads are subsequently assembled to create a full plasmid sequence. These types of results are denoted as Full sequence with the header "Addgene NGS Result." You can see the comparison between the amount of information provided by Sanger and NGS sequencing in the images below.
In our transition to NGS technology, we began by going back to the archives and getting full sequence for our many popular plasmids that didn’t already have it. Chances are, if you look at a blue flame plasmid, we’ve already had the chance to obtain NGS results! For any new plasmids that enter our doors, we’re conducting quality control using NGS.
NGS technology, although powerful, is not perfect. Like Sanger, NGS sometimes has difficulty sequencing through GC-rich regions, including the commonly used chicken beta actin (CAG) promoter. Since NGS assembles a larger sequence from many smaller sequences, regions with repeats may not assemble correctly. If we obtain an NGS result that is useful, but not 100% complete, we will still make this data available as a Partial sequence with the heading "Addgene Partial NGS Result." In some cases, there will be more information about our sequencing results in the Depositor Comments section at the bottom of the plasmid page.
As we complete the transition to NGS-based quality control, we’ll be releasing educational resources about this technology, including more blog posts and an educational tutorial. We’ll also introduce you to seqWell, our NGS sequencing partner and pioneer of novel plexWell technology. Please use the comments section below to let us know if you have any specific questions about NGS you’d like us to cover. We are also available by phone or at firstname.lastname@example.org to answer your questions about our quality control process!
Additional Resources on the Addgene Blog
- Sequencing Options for CRISPR Genotyping
- 6 Tips for Analyzing and Troubleshooting Sanger Sequencing Results
- How to Verify Your Plasmid
Resources on Addgene.org