AAV Genome Sequencing
Go from intact capsids to full-length AAV genome sequences in 3 days. Our unique library preparation process returns full unmodified AAV sequence, identifying potential issues other sequencing methods can miss.
Catch problems before they derail your project
Most AAV sequencing protocols use end-repair, annealing, amplification, and/or fragmentation steps that can generate artifacts. We skip all of that. Using a process unique to Plasmidsaurus, we can directly sequence both single stranded and double stranded DNA, preserving ITRs and genome configurations as they exist in your prep.
Verify your AAV sequence fast, with less input, and at a lower cost. You’ll get high-quality data using up to 10× less material, in 10× less time than other common long-read sequencing methods. We aim to return data within three business days of receiving samples.
Cell and gene therapy R&D
Rapidly validate early R&D transgene designs and production processes before investing in time- and money-intensive experiments. Confirm AAV genome integrity and identify problematic transgene features that may be causing truncated or snapback subgenomic populations.
Gene therapy manufacturers and CDMOs
Get better sequencing data for each lot faster and with less input so you can stay ahead of timelines and specs.
Academic labs and university core facilities
Get cost effective verification of in-house AAV production without sacrificing significant fractions of small-lot produced virus.
What depth of sequencing do I need?
Ideal for
Confirming sequence identity and detecting major structural variants as well as inverted-payload vector sequences
Variant detection threshold
Can detect variants present at >5% abundance
Coverage
~50x coverage for most abundant genome
Expected # of reads
2-5K per sample
Cost
$200
Turnaround time
3 business days from receipt
Ideal for
Detailed characterization of individual AAV samples or low complexity libraries (i.e. <10 variants)
Variant detection threshold
Can detect variants present at >1% abundance
Coverage
~200x coverage for most abundant genome
Expected # of reads
15-20K per sample
Cost
$400
Turnaround time
3 business days from receipt
Ideal for
High complexity libraries and high-resolution population profiling
Variant detection threshold
See everything
Coverage
>1000X up to millions fold coverage; reads per sample varies depending on sample number and quality
Expected # of reads
>20M per flow cell
Cost
$1750 + $200 per sample (up to 24 samples)
Turnaround time
3 business days from receipt
See what's really in your prep
It's never been so fast and easy to sequence your AAV samples. Accelerate your gene therapy workflows with the confidence of long-read sequencing.
See the whole picture
Get full-length consensus sequences, interactive genome maps, variant breakdowns, and all ITR-containing reads from just 1.0×10¹¹ vector genomes per sample.
Reveal hidden variants
De novo assemblies uncover subgenomic variants like snapback, truncated, and inverted payload genomes that reference-based methods can miss.
Move unbelievably fast
No need for complex sample prep or slow and expensive quote generation and onboarding. Get instant insights and advanced analytics delivered in three business days.
"We encountered issues with custom AAVs we purchased from a separate company and decided to try Plasmidsaurus' AAV sequencing services to determine if there were any issues with the DNA sequence. We were extremely satisfied and impressed with the quality of their service!"
Better workflow, better data
Our library preparation method directly ligates sequencing barcodes to single and double stranded DNA with a resolved ITR. We return all ITR containing reads represented in the results dataset as a downloadable FASTQ file (generally >99% of raw reads).
De novo AAV genome assembly
Plasmidsaurus’s assembly method seeks out peaks of read length then generates polished consensus assemblies similarly sized reads within that peak.
Interactive histograms
Explore read length distribution and relative abundances of reads used in de novo assemblies.
Downloadable deep-reporting
All FASTQ files used in our analysis pipeline, exact read counts per assembly, Flip-Flop orientation, FASTA and Genbank files for assemblies, and single-ITR reads (potential truncations) aligned to the longest genome are provided.
Data deliverables & bioinformatics
Consensus Sequence
Interactive Map
Assembly Quantifications (.tsv)
ITR Orientation Counts (.tsv)
Single ITR Truncation Analysis (.png)
Read Length Histogram (.png)
Virtual Gel (.png)
Raw Reads (.fastq.gz)
Ready to prep?
Ship us a minimum of 1.0 x 1011 copies of purified intact capsids. We'll handle the rest.
Related products
Whole Plasmid
Need to identify common ITR instability issues in your AAV payload plasmid? Use Whole Plasmid Sequencing to read through ITR and other high secondary-structure DNA as if it is low complexity DNA.
Amplicon
Simple setup and fast results for amplicons of any size.
Whole Genome
Fast and complete genome sequences for nearly any organism.
FAQs
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.
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.
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.
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!