title: Comparative next-generation sequencing of adeno-associated virus inverted terminal repeats.
abstract: The inverted terminal repeats (ITRs) of adeno-associated virus (AAV) are notoriously difficult to sequence owing to their high GC-content (70%) and palindromic sequences that result in the formation of a very stable, 125 bp long, T-shaped hairpin structure. Here we evaluate the performance of two widely used next-generation sequencing platforms, 454 GS FLX (Roche) and MiSeq Benchtop Sequencer (Illumina), in analyzing ITRs in comparatively sequencing linear amplification-meditated PCR (LAM-PCR) amplicons derived from AAV-concatemeric structures. While our data indicate that both platforms can sequence complete ITRs, efficiencies (MiSeq: 0.11% of sequence reads; 454: 0.02% of reads), frequencies (MiSeq: 171 full ITRs, 454: 3 full ITRs), and rates of deviation from the derived ITR consensus sequence (MiSeq: 0.8%-1.3%; 454: 0.5%) did differ. These results suggest that next-generation sequencing platforms can be used to specifically detect ITR mutations and sequence complete ITRs.
authors.affiliation: Department of Translational Oncology, National Center for Tumor Diseases (NCT) and German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Karl
authors.initials: K
authors.lastname: Petri
authors.affiliation: Department of Translational Oncology, National Center for Tumor Diseases (NCT) and German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Raffaele
authors.initials: R
authors.lastname: Fronza
authors.affiliation: Department of Translational Oncology, National Center for Tumor Diseases (NCT) and German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Richard
authors.initials: R
authors.lastname: Gabriel
authors.affiliation: Department of Translational Oncology, National Center for Tumor Diseases (NCT) and German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Christine
authors.initials: C
authors.lastname: Käppel
authors.affiliation: Department of Translational Oncology, National Center for Tumor Diseases (NCT) and German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Ali
authors.initials: A
authors.lastname: Nowrouzi
authors.affiliation: Department of Translational Oncology, National Center for Tumor Diseases (NCT) and German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: R Michael
authors.initials: RM
authors.lastname: Linden
authors.affiliation: Department of Infectious Diseases, King's College London School of Medicine, United Kingdom.
authors.forename: Els
authors.initials: E
authors.lastname: Henckaerts
authors.affiliation: Department of Translational Oncology, National Center for Tumor Diseases (NCT) and German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Manfred
authors.initials: M
authors.lastname: Schmidt
mesh_terms: D000229:Dependovirus / Q000235:genetics*; D006367:HeLa Cells / Q000821:virology; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA / Q000379:methods*; D020079:Terminal Repeat Sequences
keywords: adeno-associated virus; inverted terminal repeat; next-generation sequencing