AAV

Plasmidsaurus AAV Sequencing is performed using the newest long-read sequencing technology from Oxford Nanopore Technologies (ONT).

We extract whole AAV genomes from your intact viral capsids, and then construct an amplification-free long-read sequencing library using our unique in-house protocol to capture ITR-containing linear ssAAV or scAAV DNA.

Our approach removes the end-repair step common in other AAV-library preparation protocols. This reduces the risk of artifacts and preserves native AAV genome structure. We ligate our sequencing adaptors directly on to ITR-containing terminal sequences without other enzymatic modification or annealing. This means that we can ligate directly to native AAV genomes. We do not use thermal annealing or second strand synthesis like some other long-read AAV sequencing protocols.

We identify and assemble subspecies from the raw sequencing reads to generate high-accuracy linear consensus sequences for all detectable AAV genome subspecies (full-length, truncations, etc.) that comprise at least 1-5% of the total subspecies. We also deliver metrics on the relative quantification of each viral subspecies and histograms of genome size vs. read count.

AAV samples are sequenced without primers or amplification. Please do not ship any primers with your samples or mix primers into your samples.

We use the latest flowcells and chemistry kits from Oxford Nanopore, along with the latest super accurate basecalling model. Extensive internal testing has shown that the vast majority of our assembled AAV consensus sequences contain zero errors. Consensus accuracy tends to be above Q50, which corresponds to 99.999%, or one error per 100,000 bases.

If your AAV genomes are contained within purified, intact viral capsids (cell-free encapsulated AAV genomes in either ssAAV or scAAV genome configuration), please submit them to this AAV service!

If your AAV genomes are cloned into dsDNA circular plasmids, those can be sequenced through our Whole Plasmid sequencing service instead.

Please contact support@plasmidsaurus.com if you are interested in sequencing pre-extracted AAV DNA rather than the intact capsids that are required for this AAV service.

Yes! We return high-accuracy linear consensus sequences (.fasta) for all detectable AAV genome subspecies (isoforms) that comprise at least 1-5% of the total subspecies. We also provide the .fastq sequences of all raw reads produced by your sample.

We offer three service tiers to match different experimental needs. The more reads you request, the deeper the coverage and the more sensitivity you'll have for detecting low-abundance variants.

• Standard Service — Typical yield: 2,000–5,000 reads
   
  • Provides: ~50x coverage of the most abundant AAV genome.  
  • Can readily detect and assemble subgenomic variants present at >5% abundance of all reads (e.g. we often see snapback genomes in CAG-promoter containing samples).

  • Ideal for: Confirming sequence identity and detecting major structural variants as well as inverted-payload vector sequences.

     

• Big Service — Typical yield: 15,000–20,000 reads

  • Provides: ~200x coverage of the most abundant AAV genome.  Can detect and assemble variants present at >1% abundance.
  • Ideal for: Detailed characterization of individual AAV samples or low complexity libraries

   

• Whole Flow Cell Service — Typical yield: 20–150 million reads (15 Gb+) distributed across the number of samples submitted.
   
  • Provides: Super-deep coverage of dominant genomes (>1,000× for a 4 kb genome). Total reads per sample vary depending on sample number and quality.
  • Ideal for: High complexity libraries and high-resolution population profiling.
     

Our new chemistry requires a single stranded terminally-resolved ITR sequence to ligate our sequencing adaptors to. Therefore, host DNA and triple transfection plasmids are unlikely to be sequenced. We recommend that customers use ddPCR methods to detect and quantitate these types of contaminants.

We do detect inverted-payload vector packaging since these sequences contain ITRs.

Plasmidsaurus does offer custom sequencing services that can utilize end-repair or tagmentation based chemistries to generate reads from any and all DNA in your sample with the caveat that the advantages of our long-read sequencing of native-AAV ITR-containing DNA is lost.

Currently we are focused on sequencing engineered AAV populations containing AAV2-derived ITRs. The AAV2 ITR sequence is used in the vast majority of AAV payload plasmids.

Please note the ITR sequence used for the AAV payload transgene can be, and often is, different than the serotype of the Rep-Cap packaging plasmid.

If you are unsure of the AAV ITR sequence in your experimental design please contact support@plasmidsaurus.com and we will connect. We can accommodate other ITR configurations as custom orders. Please contact us before sending your sample!

For the Standard and Big service, we do pool samples on a flow cell but each sample is uniquely and independently barcoded. We have extensively optimized the barcoding biochemistry to prevent cross-ligation between samples and employ software filters that check for spill-over between samples before returning results. There is a very small, but non-zero, chance single reads may be returned in other samples.

The Whole Flow Cell service uses a completely fresh new flow cell, will only contain your samples, and the flow cell is never re-used after the sequencing run.

We’ve recently improved our library preparation protocol by removing the end-repair step of common AAV-library preparation protocols. If you need to run samples using the previous chemistry, contact support@plasmidsaurus.com — legacy workflows are still available by request, but may have longer turnaround times and are treated as custom orders. We strongly recommend most customers use the new service but understand that back comparison can be important for some programs.

We no longer include an initial DNase I treatment step before AAV DNA extraction because virtually all AAV production methods include an extensive upstream DNase or Benzonase treatment step.

Most likely not. Our protocol is compatible with common AAV formulation buffers as well as simple PBS or TBS. Low concentrations of Mg²⁺, glycerol, and PF-68 do not interfere with extraction or sequencing. If the sample is in a high salt solution (e.g. neutralized elution buffer from an affinity purification) we recommend buffer exchanging into something similar to PBS. 

Yes. Our workflow is compatible with relatively crude preps, including PEG-precipitated virus resuspended in PBS. As long as the sample contains at least 2×10¹¹ viral genomes, we can generate high-quality data without additional purification. See sample prep for more detailed instructions.

The most common source of a failed AAV sequencing is insufficient DNA input to our library preparation. Our ability to deliver target outputs is directly dependent on the quantity, quality, and purity of the capsids sent to us. Although there are common AAV production and purification workflows, these can vary greatly between customers and we cannot account for every possibility. 

We understand that AAV samples represent a high financial and time investment and have developed our methods to require relatively minimal sample input as well as be more robust across many sample types and AAV production/purification methods.

We quantitate all extracted AAV DNA before library preparation and if a sample falls below our recommended minimum sequencing library input we will pause the sequencing library production and contact the customer who submitted the sample. The customer can then decide to send additional material for extraction or proceed with sequencing. This will add 1-2 days to the results turnaround time. In the latter case, we cannot guarantee a minimum of returned reads or genome assembly from under-loaded samples, but the sequencing reaction may still generate usable FASTQ files for customer analysis.

If a sample fails, especially if the initial DNA extraction falls within a total yield and concentration range generally robust in our process, we will work with you to attempt to solve this problem.

If your AAV sample fails (i.e. we are not able to generate at least one consensus/assembly from your sample), you can contact us at support@plasmidsaurus.com to inquire whether the extracted yield of AAV DNA was sufficient to repeat sequencing. Please note that because we extract your entire AAV sample on the first attempt, AAV samples are typically not eligible for re-runs.