DNA QC Instructions and Information
All DNA preps are QC’d by full-length sequencing using Plasmidsaurus. Restriction digests may also be performed to confirm sequencing results.
Plasmidsaurus uses long oxford nanopore reads before generating a consensus with basecalling and polishing software giving an accuracy of TLDR: Q50 consensus accuracy = 1 error in 100,000. More can be found on the accuracy at Oxford Nanopore.
https://nanoporetech.com/accuracy
Sample preparation, shipping instructions, and cost per sample are provided on Plasmidsaurus’s website under their Prep&Ship tab.
After you submit and ship your sample using the instructions provided on the Plasmidsaurus website and your plasmid has been sequenced you will get back five files for each plasmid you submit:
- png file is a read length histogram
- fasta file is a map of the polished consensus sequence
- gbk file is similar to the fasta file but typically has more prelabled features on the plasmid map
- html file is another map of the sequenced plasmid that is generated using the pLannotate tool from the Barrick lab that annotates the plasmid map differently showing where fragments are
- stats.txt is a file of statistics generated by aligning the raw reads with Plasmidsaurus’s consensus sequence so you can see their level of confidence at each nucleotide. Plasmidsaurus can also give you the raw reads but they do not send raw reads with any sequencing results unless you specify while ordering that you want them by checking the raw reads box before submission.
You can download all of these results directly from the website to analyze yourself!
Following is a description of how we interpret the results and decide if the DNA prep is good or not. The quality and purity of the DNA we use for viral production directly impacts the quality of the viral prep we produce. This is also how we recommend you analyze your DNA preps that you intend to send to us for viral production.
It is also important to note that, because results are rarely perfect, the threshold you choose to use when deciding what is acceptable and what is not is up to you and therefore your own responsibility if you decide to QC your own DNA preps.
First step – histogram analysis
First we would take a look at the histogram provided to get an idea of the purity of the prep. In a perfect world where every DNA prep was completely pure we would see only one peak around the number of base pairs that the plasmid you are trying to amplify has. Unfortunately that is quite rare so the following figures will give you an idea of our decision making process when we are analyzing these result.
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Here we see a large peak sitting at the number of base pairs we expect this plasmid to have. Other peaks are minimal and short especially compared to the main peak. This is as close to perfect as we can typically get, this is a good clean prep.
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shows one large peak sitting at the number of base pairs we expect this plasmid to have. Other peaks are still minimal and short especially compared to the main peak but they are much more noticeable. This isn’t amazing but perfectly acceptable for us to use for transfection.
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Again, there is one large peak sitting at the number of base pairs we expect this plasmid to have. Now we are starting to see much larger peaks that aren’t our plasmid of interest. Here we would say this is ok to use if we are in need of this plasmid urgently but if we have the time and resources to remake it or the viral prep is especially sensitive we would want to try for a better result. So if you are doing your own QC and get a result like this it would be left up to your discretion if you are comfortable using this DNA prep or not.
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This is another common result that tends to toe the line of being usable or not. We have the main peak that we are looking for with one other peak that is 1/3 to 1⁄2 the size of our main peak. You could use the raw reads to decipher what exactly this second peak is and decide if the prep is usable for you from there that combined with other factors like time, material availability, or transfection/experiment sensitivity would determine if we would decide to use this prep.
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When there is a single second peak more than 1⁄2 the size of the main peak as shown in figure 5 we usually would say that this prep is not suitable for transfection. In this case when the peak is near the same size as the peak of interest Plasmidsaurus will typically send file results for both peaks meaning you will get maps for them both. This could be useful to troubleshoot what could have happened to the prep or if you believe the other plasmid present is benign in terms of what you are trying to accomplish then maybe you could decide to send this for viral prep, but do so at your own risk. We would not use this prep.
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Every so often there can be a main peak or a second peak (like in figure 6) of roughly two times the expected size of your plasmid of interest. This is likely a duplex where the plasmid of interest is doubled within itself. If this is the case and we are confident that we have a true duplex them we would use this plasmid for viral production because we have found it does not lower transfection efficiency or viral titer.
Figure 1: AAV 2/5
Figure 2: AAV 2/5
Figure 3: AAV 2/5
Figure 4: Ef1a-DIO-EYFP
Figure 5: CAG-Flex-Rev-3xGFP
figure 6 : EF1a-ChR2-YFP-WPRE-hGH
Second step – check plasmid map size and features
now we will check to see if our plasmid map is the correct size and has every part that it is expected to have. The gbk map usually has more prelabeled features so we recommend starting there. Figure 7 shows CAG-Flex-For-3xGFP and after looking at the size of the plasmid I would look for AmpR, WPRE region, CAG promotor, GOI (which is in this case 3 GFP sites), and two ITRs as those are the most critical and noticeable components to this plasmid and most AAV plasmids. If there is a substantial difference in size from the sequence results to what you expect it likely is a bad prep or after trying to find and manually label important features if they aren’t there then that is also a bad prep.
Figure 7: CAG-Flex-For-3xGFP
Third Step – align sequence with a reference
We know not every plasmid will have an already verified plasmid sequence and if you don’t have that we suggest just taking extra care to look at the features of your plasmid but if we do then we will align the sequence with the reference map we have or can find from addgene.We want and can often get a 100% match but 99.7-100% is usually a good plasmid when considering sequencing errors as shown in figure 8. Less than 99.7 you should scroll through the alignment to see where the incorrect base pairs lie and decide if this is of concern. A helpful note is if your plasmid alignment is coming up very wrong but your map and histogram are good then try flipping the DNA sequence as sometimes snapgene has them reversed. Also remember that it is more common for sequencing errors to occur in C-G pairings as they are harder to separate so if there is a string of C-G pairs that are incorrect compared to your reference map then its possible that it was a sequencing error and not your actual plasmid.
Figure 8: CAG-Flex-For-3xGFP
Snapgene has tutorials and instructions on how to do all of techniques that we use like flipping sequences, searching for features, aligning two files, ect...
This is a fairly comprehensive description of our QC process but if you have any more questions feel free to reach out!
Page Written by Aaliyah O’Dell, Research Technician