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Frequently Asked Questions (FAQ)
Gene therapy is a method of treating genetic disease by adding, deleting, or correcting genetic material in the cells of the person with a disease with the goal to change the course of disease.
Adeno-associated virus (AAV) is a non-enveloped virus approximately 4.7 kilobases (kb) in size and can be engineered to deliver DNA to target cells. AAV vectors are typically preferred due to low toxicity, dependence upon other viruses for replication, broad tropism and the ability to infect both dividing and non-dividing cells.
Read more: "PacBio Gene Therapy + Gene Editing"
PacBio AAV sequencing offers a distinct advantage of being able to sequence entire, intact, ~4.7kb construct with high accuracy. The ability to see the full-length vector genome sequence in a single, continuous read is important for discovery and for identifying truncated products while high accuracy is important for identifying undesired mutations.
Read more: "PacBio Gene Therapy + Gene Editing"
Once your library is prepared, a PacBio instrument such as the Sequel II system or the Revio can be used to sequence it. PacBio's Single Molecule Real Time (SMRT) sequencing uses millions of tiny wells called zero-mode waveguides (ZMWs). Single molecules of rAAV DNA are immobilized in these wells, and as the polymerase incorporates each nucleotide, light is emitted, and nucleotide incorporation is measured in real time. The reactions are recorded in a format that can then be analyzed using on-instrument tools, as well as third-party and open-source software such as LAAVA.
Read more: "PacBio Sequencing 101: from DNA to discovery — the steps of SMRT sequencing"
Currently there is a library preparation limitation on the ability to detect certain configurations of self-complementary AAVs, where truncated genomes do not fold into the blunt-end dsDNA formation required for SMRTbell hairpin adapter ligation.
PacBio bias can be observed on specific fragment lengths. These biases can be measured with spike-in sequences of known quantities.
Long-read sequencing obtains the complete nucleotide sequences of the viral genomes packaged in an AAV product sample, including full-length viral genomes and contaminants such as truncated genomes, chimeric molecules, host cell genomes, and other plasmids. From this data, further computational analysis can determine the frequency of reads from each source (e.g. vector, backbone, chimeric, helper, repcap, host), distribution of read data by type and subtype (ssAAV/scAAV, full/partial, read-through/reverse packaging, other contaminants), variants by type and position (mismatch, insertion, deletion), and ITR configurations (flip/flop).
PacBio long reads sequencing enables identification of reads lengths and positions. Read end counts can be used to identify truncation hotspots.
Snapback genomes (SBGs) are partial vector genomes with the left or right moiety of standard duplex rAAV genomes. SBGs can be classified as symmetric, where the top and bottom strands complement each other, or asymmetric, where DNA at the bottom strand does not match the top strand completely and therefore promotes loop formation in the middle region.
- In a single-stranded AAV genome, these manifest in the sequencing data as self-complementary genomes where reads map on both polarities of the vector.
- In a self-complementary genome, the manifestation will be self-complementary left or right partial where reads are partially mapping to the vector.
Read-through genomes are characterized by the encapsidation of DNA that extend beyond the ITR and into the backbone sequence. Reverse-packaged genomes are packaged from ITR-to-ITR but encompass sequences that exclusively span the backbone.
In the computational analysis, sequencing reads that align to both polarities, forward and reverse complement, are classified as "self-complementary" even if the vector design is single-stranded. These vector genomes can occur when there is template switching during replication, for example snapback genomes (above).
One ITR is formed by two palindromic arms, called B–B' and C–C', embedded in a larger one, A–A'. The order of the two palindromic sequences within an ITR defines its "flip" or "flop" orientation. In a typical sample, four ITR configurations are expected to be observed in equal proportion: (left-right) flip-flip, flip-flop, flop-flip, and flop-flop. These proportions can be a useful QC criterion for AAV.
There is no clear guidance from the FDA in terms of threshold. As a best practice, we consider some contamination threshold as well as the amount of full AAVs detected.
The PacBio on-instrument AAV mode generates HiFi reads for proper handling of scAAV and ssAAV structures.
Reads that are longer than ~5 kb, too large to be packaged, will be detected and this should be at a very low frequency. That can be associated to sequencing issues and library preparation issues such as DNA hybridization.