A Look at Addgene's QC Process

Nobody’s perfect! This is a platitude I’m sure we have all heard. It’s a truth that extends to both people and the tools we use. Scientists utilize an array of tools that rely on DNA amplification and manipulation to produce and edit plasmids. These are prone to error — both human and biological. Even the most high-fidelity polymerases can make mistakes and lead to the accumulation of errors in DNA sequences. In research, these errors can affect experimental processes, results, and subsequent conclusions. Being vigilant about these errors is an important part of maintaining the quality of reagents, especially shared ones. 

The importance of QC

Plasmids are a fundamental part of research. They are easy to produce, easy to edit, and easy to share. However, the cloning process — including the propagation of plasmids in bacteria — has many points at which small errors can be introduced. Eventually, these errors could cause the plasmid to stop functioning properly or be something completely different than what was originally intended. Sharing unverified plasmids can lead to far reaching consequences, from wasting time and money on optimizing experiments, to false conclusions. 

Figure 1. Potential points where DNA errors can be introduced, indicated by exclamation marks. Error points include places where DNA is being created, such as PCR amplification or bacterial growth. Created with BioRender.com.

At Addgene, we put a lot of effort into our quality control (QC) processes to screen for and reduce these errors. These processes allow us to have a high standard of sequence quality across all plasmids in our collection. This standardization provides security for requestors, ensuring that the plasmid they ordered contains the correct inserts, tags, promoters, and other critical functional components.

Fun fact! Some older plasmids in our collection do not have a full sequence, as they were deposited before 2017 when Addgene started sequencing all incoming plasmids. If you are interested in requesting one of these plasmids, you can request that it be fully sequenced by Addgene’s QC team. Email help@addgene.org to get started!

Addgene’s QC process

Addgene prioritizes the sequence quality of the plasmids we distribute to scientists worldwide. Depositing scientists provide different types of data for each of their plasmids, from descriptions of key components of the plasmids to partial, full, or annotated plasmid sequences. These sequences can also be confirmed or theoretical. No matter the level of information that is provided, each deposit goes through the same QC process to ensure all plasmids pass our high-quality standards and contain the expected plasmid features. 

Figure 2. Addgene’s basic QC process. Created with BioRender.com.

Once Addgene receives deposited plasmids, they undergo complete plasmid sequencing using an in-house Illumina MiSeq. The raw sequencing reads are assembled into a circularized plasmid sequence de novo (without the use of a reference sequence) using a bioinformatics pipeline developed at Addgene. The assembled plasmid sequence is then compared to the information provided by the depositor.

If a depositor provides a sequence for the plasmid, QC scientists will first align the de novo NGS result to the deposited sequence(s) in SnapGene. SnapGene annotates the plasmid maps based on SnapGene’s “Common Feature” library — with some additional features added by the QC team — and allows QC scientists to view all open reading frames. If a deposited sequence is not available, QC scientists will compare the NGS results to the plasmid information, other plasmids in the deposit, NCBI sequence and protein data, as well as available backbone sequences. All of these resources allow QC scientists to easily visualize and identify sequence differences between our NGS results and deposited sequences and plasmid information. 

For each plasmid deposited at Addgene, QC scientists confirm: 

1. The size of the plasmid to ensure our assembly result is accurate
2. The gene/insert or any major feature highlighted by the deposited scientist 
3. The backbone elements based on the information provided and vector type 
4. Any tags, selectable markers, and fusion proteins listed for each plasmid 

If the plasmids pass all inspection points, they are made available for request. Otherwise, they continue through the QC pipeline to resolve any issues. 

Catching and addressing QC issues

Addgene’s QC team has developed a process for identifying, categorizing, and resolving a variety of QC issues. This process ensures that any potential sequence discrepancies can be caught prior to distribution. Catching these errors prevents future experimental issues, saving time and money for scientists.

What is a QC issue?

At Addgene, a QC issue is defined as a discrepancy that we believe may affect the function of the plasmid. This includes point mutations, frame shifts, missing elements, and nucleotide indels or mismatches in significant plasmid elements. Which differences are considered a QC issue is reviewed yearly at a best practices meeting, where the QC team updates and reviews our sequencing QC standards to ensure the entire team is using the same rubric and making the same decisions when analyzing the wide variety of discrepancies encountered in plasmids deposited to Addgene. This review process also highlights other QC topics that may require additional attention during analysis. These include topics such as: locations in a plasmid that are less likely to affect function when mutated; elements that are difficult to sequence; approaches to confirm plasmid sequences without a reference; and identifying mixed plasmid and nucleotide populations. All of these topics require additional considerations to resolve.

Sequencing and assembly issues

Sequence discrepancies are not always a result of mutations in the plasmid, but can arise due to errors in the sequencing and assembly pipeline. There are regions of plasmids that are particularly difficult to sequence and assemble using the Illumina MiSeq platform, such as GC-rich regions and highly repetitive sequences and/or features. Most of these regions are identified during the QC process, following sequencing and assembly.

In cases where the QC team suspects an assembly issue, they manually revisit the raw NGS reads and reassemble using a program called Geneious. Here, the raw reads are mapped against a reference sequence, if one was provided. If no reference is given, de novo assembly in Geneious can still provide additional insights to resolve the assembly and determine if there are any notable QC issues. Mapping the raw NGS reads to a reference sequence can also help QC scientists determine if enough reads were acquired from the MiSeq for assembly or with enough coverage over plasmid regions to confirm their presence with confidence.

If this approach does not resolve the identified differences, the plasmids are sent out to Plasmidsaurus for long-read nanopore sequencing. This platform sequences the entire plasmid in one read and thus can resolve sequencing and assembly issues that arise due to the short 200–250 base pair reads sequenced on the Illumina MiSeq. 

Resolving QC issues

If the QC team identifies a QC issue, they will contact the depositing lab to review the discrepancies. Often, the identified issues will not affect function and the plasmid can pass through QC and be made available for distribution. In these cases, the QC team leaves a note on the plasmid page to make requesting scientists aware of the identified discrepancy in the plasmid, and that it is not known to impact the function of the plasmid. If the issue is likely to affect plasmid function, we request the lab provide a new, sequence-verified replacement for the plasmid, and the full QC process is started over again after receiving the replacement plasmid at Addgene.

QC is a team effort

Addgene’s QC team is full of dedicated scientists with expertise in sequencing technologies and bioinformatics. Through their efforts, the QC team finds errors in 30% of deposited plasmids! We are committed to ensuring that we maintain a high standard of sequence accuracy across all of our plasmids and provide transparency in sequence discrepancies. While Addgene’s QC process adds time to the deposit process, we believe this to be non-negotiable. By maintaining a high standard of quality control, we have built a trusted plasmid catalog containing over 150,000 sequence-verified constructs to share with scientists across the globe.

This post was written by Alyssa Shepard, with significant contributions from Alyssa Cecchetelli, Cary Valley, and Holly McQueary.

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Resources

Additional resources on the Addgene blog

• Addgene's Tips for Plasmid Quality Control
• Plasmids 101: In-house NGS Plasmid Quality Control Now at Addgene
• Bioinformatics at Addgene

Resources on Addgene.org

• Educational Resources
• Sequence Analysis of a Plasmid Protocol