Single-Stranded DNA Virus Sequencing (SSV-Seq) for Characterization of Residual DNA and AAV Vector Genomes

Methods Mol Biol. 2019;1950:85-106. doi: 10.1007/978-1-4939-9139-6_5.


With the success of clinical trials using recombinant adeno-associated viral vectors (rAAV), regulatory agencies ask for a more comprehensive characterization of process- and product- related impurities found in rAAV stocks in order to assess the potential risks for patients. During production, rAAV capsids are known to internalize illegitimate DNA fragments in addition to their recombinant genome. These contaminants can come from plasmid or helper virus DNA as well as from the producer host cell. Here, we describe a method based on high-throughput sequencing to identify and quantify residual DNA in rAAV vector lots. Contrary to qPCR, SSV-Seq (Single-Stranded DNA Virus Sequencing) offers a nonselective approach to determine the percentage of each DNA contaminant and analyze rAAV vector genome identity.

Keywords: AAV; Identity; Quality control; Residual DNA; Sequencing; Viral vectors.

MeSH terms

  • Computational Biology / methods
  • DNA, Single-Stranded*
  • DNA, Viral*
  • Dependovirus / genetics*
  • Genetic Vectors / genetics*
  • Genome, Viral*
  • High-Throughput Nucleotide Sequencing
  • Sequence Analysis, DNA*
  • Software
  • Workflow


  • DNA, Single-Stranded
  • DNA, Viral