Genome concentration, characterization, and integrity analysis of recombinant adeno-associated viral vectors using droplet digital PCR

Precise, reproducible characterization of AAV is critical for comparing preclinical results between laboratories and determining a safe and effective clinical dose for gene therapy applications. In this study, we systematically evaluated numerous parameters to produce a simple and robust ddPCR protocol for AAV characterization. The protocol uses a low ionic strength buffer containing Pluronic-F68 and polyadenylic acid to dilute the AAV into the ddPCR concentration range and a 10-minute thermal capsid lysis prior to assembling ddPCR reactions containing MspI. A critical finding is that the buffer composition affected the ITR concentration of AAV but not the ITR concentration of a double stranded plasmid, which has implications when using a theoretical, stoichiometric conversion factor to obtain the titer based on the ITR concentration. Using this protocol, a more comprehensive analysis of an AAV vector formulation was demonstrated with multiple ddPCR assays distributed throughout the AAV vector genome. These assays amplify the ITR, regulatory elements, and eGFP transgene to provide a more confident estimate of the vector genome concentration and a high-resolution characterization of the vector genome identity. Additionally, we compared two methods of genome integrity analysis for three control sample types at eight different concentrations for each sample. The genome integrity was independent of sample concentration and the expected values were obtained when integrity was determined based on the excess number of positive droplets relative to the number of double positive droplets expected by chance co-encapsulation of two DNA targets. The genome integrity was highly variable and produced unexpected values when the double positive droplet percentage was used to calculate the genome integrity. A protocol using a one-minute thermal capsid lysis prior to assembling ddPCR reactions lacking a restriction enzyme used the non-ITR assays in a duplex ddPCR milepost experiment to determine the genome integrity using linkage analysis.


Genome concentration calculations
A sample with a genome concentration of 50,000 copies/µL at capsid lysis will produce a ddPCR reaction with 2,500 copies/µL when 1 µL of the sample is used to prepare a 20 µL reaction (i.e., a 1:20 dilution). So, the genome concentration of the sample at capsid lysis is: Mix all components of the master mix thoroughly in a DNA LoBind tube and aliquot 45 µL into two tubes of an 8-tube PCR strip. Add 5 µL of a viral sample to each tube, cap the tubes, mix thoroughly, pulse in a PCR tube centrifuge, and incubate for 30 minutes at 37°C using a C1000 Touch thermal cycler.

Example master mix preparation for genome characterization
For one genome characterization with 6 singleplex assays in duplicate, make enough master mix for three reactions with each assay (duplicate + 1 extra). Calculate the number of reactions needed (6 assays × 3 = 18), add 10% (~2) to account for fluid loss to solid surfaces for a total of 20 reactions. If the calculated number of reactions is not a multiple of 4, round up to the next multiple of 4 for convenient pipetting of 5 U MspI per reaction at 20 U/µL.
If the required number of duplicate reactions is not a multiple of eight (i.e., the number needed for a fully loaded droplet generation cartridge), make blank wells by mixing equal amounts of 2× supermix and nuclease-free water, not DEPC-treated (ThermoFisher, AM9937). In this case with 6 assays in duplicate, 12 sample wells and 4 blank wells are needed, so make enough for 5 blank wells by mixing 50 µL of 2× supermix with 50 µL of water. Add all master mix components to a DNA LoBind microcentrifuge tube, mix thoroughly by vortexing and pulse centrifuge. Aliquot 57 µL of the master mix into individual DNA LoBind tubes. Add 3 µL of the appropriate 20× FAM assay to each tube, thoroughly vortex mix and pulse centrifuge. Prepare droplets using two replicates from each assay, thermal cycle, and read droplets.

Number of ddPCR reactions needed for a genome characterization using concentration ratios
Concentration ratios can be determined using singleplex or duplex data. For singleplex data, the number of reactions needed (n) is equal to the number of assays. All pairwise concentration ratios can be calculated from the singleplex data. For duplex data, the number of unique pairwise combinations of n assays is n(n-1)/2. Therefore, fewer ddPCR singleplex reactions are needed to analyze all pairwise concentration ratios when more than three assays are used for genome characterization.

Linkage analysis
Linkage analysis is a relatively unexplored application of droplet digital PCR that has great potential to phase genomic variants or characterize the integrity of a DNA molecule. 12-17 The simplest experiment for linkage analysis uses a duplex ddPCR reaction that contains a FAM assay and a HEX assay designed to amplify two target sequences. If the two target sequences are on the same DNA molecule, the two-dimensional (2D) fluorescence plot will contain more double positive droplets than would be expected from random colocalization into droplets of the two independent target sequences, which are located on separate DNA molecules. A more complex linkage experiment uses a milepost strategy to determine linkage as a function of assay separation. 17 This experiment uses a series of duplex ddPCR reactions that contain an anchor assay in FAM, for example, that is duplexed with different HEX assays that are increasingly further away from the anchor sequence on the DNA molecule.
The easiest way to understand the effect of linkage is in the regime when no double positive droplets are expected by chance encapsulation of the target sequences for both the FAM assay and HEX assay. In this situation, all double positive droplets are the result of physical linkage between the two assays.
Consider the situation of a single copy target for both the FAM and HEX assay on the same DNA molecule.
The FAM target sequence is represented by the blue rectangle and the HEX target sequence is represented by the green rectangle. Also, assume that digestion with a restriction enzyme prior to droplet formation will separate the two target sequences so that they partition independently into droplets.
When there are 10 DNA molecules in a ddPCR reaction, the two-dimensional (2D) fluorescence plot will look like when the DNA molecules are restriction digested prior to droplet formation. There will be 10 single positive droplets containing the FAM target sequence (blue), 10 single positive droplets containing the HEX target sequence (green), and no double positive droplets (orange) containing both target sequences. The linkage concentration in copies/µL is automatically calculated by the ddPCR software for each well in any duplex or multiplex experiment and is available in the data table. For this particular example with independently segregating target sequences at a low concentration, there are no double positive droplets expected and the linkage concentration is zero.
If there is no restriction digestion of the DNA prior to droplet formation, the two target sequences will be on the same DNA molecule and will partition into the same droplets. As a result, the droplets will be double positive for both the FAM and HEX target sequences and the 2D fluorescence plot will look like with 10 double positive droplets (orange) containing both sequences and no single positive droplets containing either the FAM (blue) or HEX (green) target sequence. In this case, zero double positive droplets are expected due to chance encapsulation but linkage between the two target sequences produces all 10 of the double positive droplets. The linkage percentage based on the average concentration of the FAM and HEX assay assumes that the concentration of the two targets will be similar. However, it is possible that the FAM and HEX assay concentrations may be different for two single copy targets due to molecular sampling, differential target accessibility, dissimilar amplicon sizes, or variable DNA fragmentation. For the previous example, assume that the FAM assay does not amplify in one droplet due to target accessibility. Instead of being a double positive (orange) droplet, the droplet that did not amplify the FAM target is now a single positive HEX (green) droplet even though it contains a DNA molecule that contains the target sequence for both the FAM and HEX assays. The 2D fluorescence plot will look like: where the FAM assay concentration is underestimated and the HEX assay concentration is unaffected. In this case where the sample is highly linked and there is molecular dropout of one assay, the linkage can be calculated using the following empirical equations that compensate for differences in assay concentration: [