AAV Genome Sequencing
| Sample Type | Category | Size | Concentration | Minimum Volume | Price Per Sample (USD) |
|---|---|---|---|---|---|
| AAV | Standard | See Instructions Below | $200 | ||
Description of Service
Recently updated! Just released, our new ITR-to-ITR AAV Genome Sequencing represents a
significant improvment over alternative AAV sequencing methods. We developed a new sequencing approach that selects for native
ITR-containing DNA with no end-repair and minimal enzymatic manipulation. The result is a more accurate representation of genomes,
whole and truncated, in your sample.
This new service is compatible with all common AAV genome configurations, e.g. self-complimentary and standard single-stranded.
All types of AAVs can be submitted in one batch order.
Coming Soon! New and improved analysis tools for rapid evalution of results.
If you have run previous samples with the original method and require a direct cross-comparison please contact us to
process your samples using the original protocol.
The AAV Genome Sequencing service is intended for the full-length sequencing of whole AAV genomes, using purified (cell-free), intact AAV viral capsids as input. This service is compatible with both single-stranded (ss) and self-complementary (sc) AAV genome configurations, including those that contain hairpin structures and ITRs.
In the vast majority of cases, we deliver AAV sequencing results within 3 business days of receipt of your samples.
This service is performed using the newest long-read sequencing technology from Oxford Nanopore Technologies, and includes the following components:
-
Extracting whole AAV genomes from your intact viral capsids.
- Note: 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.
- Constructing an amplification-free, end-repair free, long-read sequencing library that enriches for ITR-containing sequences using the newest v14 library prep chemistry.
- Sequencing the library with a primer-free protocol using the most accurate R10.4.1 flow cells (all the raw data produced by your sample is delivered in .fastq format).
- Identifying and assembling 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, depending on the sample. We also deliver metrics on the relative quantification of each viral subspecies and histograms of genome size vs. read count.
Figure 1. Diagram of AAV data analysis workflow.
Figure 2. Examples of AAV samples with different genome configurations and isoform profiles. Sample A: High quality ssAAV, indicating the single expected full-length isoform at 4,484 bp. Sample B: ssAAV with the expected full-length isoform at 3,275 bp, in addition to multiple snapback genome isoforms (truncation products) and a heterogeneous unassembled peak. Sample C: scAAV with the expected full-length isoform at 3,682 bp, in addition to the uncomplemented scAAV half-product. Sample D: scAAV with the expected full-length isoform at 4,443 bp, in addition to the uncomplemented scAAV half-product and multiple snapback genome isoforms (truncation products).
The input required for this service is 1.0 x 1011 total genome copies (GC) of purified (cell-free), intact AAV viral capsids, in a volume of 200 µl of PBS (viral titer = 5.0 x 108 GC/μl).
Please contact support@plasmidsaurus.com if you are interested in sequencing pre-extracted AAV DNA rather than the required intact capsids. If your AAV genomes are cloned into dsDNA circular plasmids, those can be sequenced through our Whole Plasmid sequencing service!
Please ship purified AAV samples in 200 μl strip tubes that are tightly closed, lids wrapped with parafilm, and placed inside a 50 ml conical tube to protect against leakage during shipping. Please label your strip tubes per our Shipping Instructions.
Data & File Types Delivered
When your results are ready, you will receive an email notification. Once you sign in to your account, you can download these results from your Dashboard. More detailed information about data & file types can be found in the Results Interpretation Guides.
- Consensus sequence (.fasta file): Provides 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, depending on the sample.
- Read length histogram (.png file): Displays the read length distribution of all the raw reads produced by your sample, with read length (genome size) vs. number of reads (molecular counts).
- Isoform quantifications (.tsv): Indicates relative quantification of each AAV genome subspecies (isoform) as a fraction of total reads obtained.
- Raw read sequences (.fastq.gz file): Provides the sequences of all raw reads produced by your sample. Please note that returning all raw reads means there is a small chance of demultiplexing error, so a few reads from your sample might be returned to another customer on the same sequencing run.
Our ability to deliver these target outputs is directly dependent on the quantity, quality, and purity of the viral capsids sent to us, so we do not guarantee results.
If your 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 reruns.
Preparing Your AAV Samples
The input required for this service is 1.0 x 1011 total genome copies (GC) of purified (cell-free), intact AAV viral capsids, in a volume of 200 µl of PBS (viral titer = 5.0 x 108 GC/μl). This service is compatible with both single-stranded (ss) and self-complementary (sc) AAV genome configurations, including those that contain hairpin structures and ITRs. This service is NOT compatible with AAV genomes that are cloned into dsDNA circular plasmids (which should be submitted instead to our Whole Plasmid sequencing service).
AAV samples are sequenced WITHOUT primers, so please DO NOT ship any primers with your samples or mixed into your samples.
Due to customs regulations, the AAV genome sequencing service is currently only available to US customers.
Our low sequencing prices and fast turnaround times rely on researchers to perform full QC to ensure that AAV capsid samples meet the following requirements prior to shipping:
A commonly used method for AAV capsid purification is ultracentrifugation with a cesium chloride (CsCl) density gradient or iodixanol gradient (IOD) (see protocols in Strobel et al, 2015). Please refer to published literature for additional capsid purification protocols.
For best results, please verify that purified AAV samples contain no remaining host cells or cell lysate, and ship the final purified capsids in PBS. Our viral capsid extraction method includes a DNase pre-treatment to deplete any non-encapsulated DNA before sequencing.
After submitting your AAV order through your Dashboard, you will be asked to edit the “Notes” field to specify how you purified your sample.
A commonly used method for AAV capsid quantification (titration) is qPCR or digital droplet PCR (ddPCR) (see protocol from Addgene). Please refer to published literature for additional capsid titration protocols.
The input required for this service is 1.0 x 1011 total genome copies (GC) of purified (cell-free), intact AAV viral capsids, in a volume of 200 µl of PBS (viral titer = 5.0 x 108 GC/μl). Please normalize your samples to this specific titer.
Please ship purified AAV samples in 200 μl strip tubes that are tightly closed, lids wrapped with parafilm, and placed inside a 50 ml conical tube to protect against leakage during shipping. Please label your strip tubes per our Shipping Instructions.
Ship your purified AAV samples directly to our Oregon lab (address provided when placing an order in your Dashboard), in a styrofoam box with an ice pack to keep the samples chilled in transit. Please DO NOT ship your AAV samples at room temperature through a Plasmidsaurus dropbox.
Interpreting Your Results
Please refer to our Results Interpretation Guides for details.