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Thorough molecular configuration analysis of noncanonical AAV genomes in AAV vector preparations
Zhang, Junping Yu, Xiangping Chrzanowski, Matthew Tian, Jiahe Pouchnik, Derek Guo, Ping Herzog, Roland W. Xiao, Weidong
Zhang, Junping
Yu, Xiangping
Chrzanowski, Matthew
Tian, Jiahe
Pouchnik, Derek
Guo, Ping
Herzog, Roland W.
Xiao, Weidong
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2024-02-18
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https://doi.org/10.1016/j.omtm.2024.101215
Abstract
The unique palindromic inverted terminal repeats (ITRs) and single-stranded nature of adeno-associated virus (AAV) DNA are major hurdles to current sequencing technologies. Due to these characteristics, sequencing noncanonical AAV genomes present in AAV vector preparations remains challenging. To address this limitation, we developed thorough molecule configuration analysis of noncanonical AAV genomes (TMCA-AAV-seq). TMCA-AAV-seq takes advantage of the documented AAV packaging mechanism in which encapsidation initiates from its 3′ ITR, for AAV-seq library construction. Any AAV genome with a 3′ ITR is converted to a template suitable to adapter addition by a Bst DNA polymerase-mediated extension reaction. This extension reaction helps fix ITR heterogeneity in the AAV population and allows efficient adapter addition to even noncanonical AAV genomes. The resulting library maintains the original AAV genome configurations without introducing undesired changes. Subsequently, long-read sequencing can be performed by the Pacific Biosciences (PacBio) single-molecule, real-time (SMRT) sequencing technology platform. Finally, through comprehensive data analysis, we can recover canonical, noncanonical AAV DNA, and non-AAV vector DNA sequences, along with their molecular configurations. Our method is a robust tool for profiling thorough AAV-population genomes. TMCA-AAVseq can be further extended to all parvoviruses and their derivative vectors.
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Junping Zhang, Xiangping Yu, Matthew Chrzanowski, Jiahe Tian, Derek Pouchnik, Ping Guo, Roland W. Herzog, Weidong Xiao, Thorough molecular configuration analysis of noncanonical AAV genomes in AAV vector preparations, Molecular Therapy - Methods & Clinical Development,
Volume 32, Issue 1, 2024, 101215, ISSN 2329-0501, https://doi.org/10.1016/j.omtm.2024.101215. (https://www.sciencedirect.com/science/article/pii/S2329050124000317)
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Elsevier
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Molecular Therapy, Vol. 32, Iss. 1
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