Recent successful clinical trials with recombinant adeno-associated viral vectors (rAAVs) have led to a renewed interest in gene therapy. However, despite extensive developments to improve vector-manufacturing processes, undesirable DNA contaminants in rAAV preparations remain a major safety concern. Indeed, the presence of DNA fragments containing antibiotic resistance genes, wild-type AAV, and packaging cell genomes has been found in previous studies using quantitative polymerase chain reaction (qPCR) analyses. However, because qPCR only provides a partial view of the DNA molecules in rAAV preparations, we developed a method based on next-generation sequencing (NGS) to extensively characterize single-stranded DNA virus preparations (SSV-Seq). In order to validate SSV-Seq, we analyzed three rAAV vector preparations produced by transient transfection of mammalian cells. Our data were consistent with qPCR results and showed a quasi-random distribution of contaminants originating from the packaging cells genome. Finally, we found single-nucleotide variants (SNVs) along the vector genome but no evidence of large deletions. Altogether, SSV-Seq could provide a characterization of DNA contaminants and a map of the rAAV genome with unprecedented resolution and exhaustiveness. We expect SSV-Seq to pave the way for a new generation of quality controls, guiding process development toward rAAV preparations of higher potency and with improved safety profiles.
Sample Corespondance
Table indicating the correspondence between samples and IDs
Chromosomal_distribution
Read count in human chromosomes used for Figure 3
References
Fasta and Genbank files containing the sequences and annotations for the references non-available in public repositories (Ad5_in_293_genome, Backbone-AAV-CMV-GFP-hTK, Cassette_AAV-CMV-GFP-hTK, pDP8_Kana, SSV9K2-CMV-GFP-HygroTK-bGHpA)
Variants
VCF files and alternative allele counting files along rAAV genome, used for Figure 4 b.
Coverage
BED files containing the base per base sequencing coverage along rAAV genome + Summary table with normalized data. Used for Figure 4 a.
Circos_Data
Data and configuration file used to generate the Circos plot. Used for figure 5
ContaVect_Configuration_Files
Configuration files for each sample used to run ContaVect
RUN1_S4_R1.fastq
Raw fastq file containing data for sample id RUN1_S4 R1
RUN1_S1_R1.fastq.gz
RUN1_S4_R2.fastq
Raw fastq file containing data for sample id RUN1_S4 R2
RUN1_S1_R1.fastq
Raw fastq file containing data for sample id RUN1_S1 R1
RUN1_S1_R2.fastq
Raw fastq file containing data for sample id RUN1_S1 R2
RUN1_S2_R1.fastq
Raw fastq file containing data for sample id RUN1_S2 R1
RUN1_S2_R2.fastq
Raw fastq file containing data for sample id RUN1_S2 R2
RUN1_S3_R1.fastq
Raw fastq file containing data for sample id RUN1_S3 R1
RUN1_S3_R2.fastq
Raw fastq file containing data for sample id RUN1_S3 R2
RUN1_S5_R1.fastq
Raw fastq file containing data for sample id RUN1_S5 R1
RUN1_S5_R2.fastq
Raw fastq file containing data for sample id RUN1_S5 R2
RUN1_S6_R1.fastq
Raw fastq file containing data for sample id RUN1_S6 R1
RUN1_S6_R2.fastq
Raw fastq file containing data for sample id RUN1_S6 R2
RUN1_S7_R1.fastq
Raw fastq file containing data for sample id RUN1_S7 R1
RUN1_S7_R2.fastq
Raw fastq file containing data for sample id RUN1_S7 R2
RUN1_S8_R1.fastq
Raw fastq file containing data for sample id RUN1_S8 R1
RUN1_S8_R2.fastq
Raw fastq file containing data for sample id RUN1_S8 R2
RUN2_S1_R1.fastq
Raw fastq file containing data for sample id RUN2_S1 R1
RUN2_S1_R2.fastq
Raw fastq file containing data for sample id RUN2_S1 R2
RUN2_S2_R1.fastq
Raw fastq file containing data for sample id RUN2_S2 R1
RUN2_S2_R2.fastq
Raw fastq file containing data for sample id RUN2_S2 R2
RUN2_S3_R1.fastq
Raw fastq file containing data for sample id RUN2_S3 R1
RUN2_S3_R2.fastq
Raw fastq file containing data for sample id RUN2_S3 R2
RUN2_S4_R1.fastq
Raw fastq file containing data for sample id RUN2_S4 R1
RUN2_S4_R2.fastq
Raw fastq file containing data for sample id RUN2_S4 R2
RUN2_S5_R1.fastq
Raw fastq file containing data for sample id RUN2_S5 R1
RUN2_S5_R2.fastq
Raw fastq file containing data for sample id RUN2_S5 R2
RUN2_S6_R1.fastq
Raw fastq file containing data for sample id RUN2_S6 R1
RUN2_S6_R2.fastq
Raw fastq file containing data for sample id RUN2_S6 R2
RUN2_S7_R1.fastq
Raw fastq file containing data for sample id RUN2_S7 R1
RUN2_S7_R2.fastq
Raw fastq file containing data for sample id RUN2_S7 R2
RUN2_S8_R1.fastq
Raw fastq file containing data for sample id RUN2_S8 R1
RUN2_S8_R2.fastq
Raw fastq file containing data for sample id RUN2_S8 R2