Recombinant adeno-associated virus (rAAV) vectors are increasingly popular gene delivery tools in biological systems. They are safe and lead to high-level, long-term transgene expression. rAAV are available in multiple serotypes, natural or engineered, which enable targeting to a wide array of tissues and cell types. In addition, rAAVs are relatively easily produced in a well-equipped lab or obtained from a viral vector core facility. Unfortunately, there is no standardization of quality control assays beyond titering and purity assessments. Next-generation sequencing (NGS) can be used to identify rAAV preparations. Because the rAAV genome is single stranded, previous studies have assumed that rAAV genomes must be converted to double strands before NGS. We demonstrate that rAAV DNA extracts exist primarily as double-stranded species. We hypothesize that these molecules form from the natural base pairing of complementary [+] and [−] strands after DNA extraction and show that rAAV DNA extracts are sufficient templates for downstream NGS without the labor-intensive double-stranding step. Here, we provide a detailed protocol for the simple and rapid NGS of rAAV genomes from DNA extracts. With this protocol, users can quickly confirm the identity of an rAAV preparation and detect the presence of contaminating rAAV DNA. In addition, we share custom Python scripts that allow users to accurately determine the serotype and detect Cre-independent DNA recombination events in rAAV containing Lox sites within minutes. We have used these scripts to analyze more than 100 rAAV preparations. Although we focused on the detection of cross-contaminating rAAV DNA and recombination events, our Python scripts can be customized to detect other sequences or events, such as reverse packaging of plasmid backbone or DNA from the packaging cell line. We find that the NGS of rAAV DNA extracts, termed viral genome sequencing, is a simple and powerful method for rAAV validation.

中文翻译：

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一种新型的下一代测序和分析平台，用于评估来自病毒 DNA 提取物的重组腺相关病毒制剂的身份。

重组腺相关病毒 (rAAV) 载体是生物系统中越来越流行的基因传递工具。它们是安全的，可实现高水平、长期的转基因表达。rAAV 有多种血清型，天然的或工程化的，可以靶向多种组织和细胞类型。此外，rAAV 相对容易在设备齐全的实验室中生产或从病毒载体核心设施中获得。不幸的是，除了滴定和纯度评估之外，没有质量控制检测的标准化。下一代测序 (NGS) 可用于识别 rAAV 制剂。由于 rAAV 基因组是单链的，以前的研究假设 rAAV 基因组必须在 NGS 之前转换为双链。我们证明 rAAV DNA 提取物主要作为双链物种存在。我们假设这些分子由 DNA 提取后互补 [+] 和 [-] 链的自然碱基配对形成，并表明 rAAV DNA 提取物是下游 NGS 的足够模板，无需劳动密集型双链步骤。在这里，我们提供了一个详细的协议，用于从 DNA 提取物中简单快速地对 rAAV 基因组进行 NGS。使用此协议，用户可以快速确认 rAAV 制剂的身份并检测污染 rAAV DNA 的存在。此外，我们共享自定义 Python 脚本，允许用户在几分钟内准确确定血清型，并在包含 Lox 位点的 rAAV 中检测独立于 Cre 的 DNA 重组事件。我们已经使用这些脚本分析了 100 多种 rAAV 制剂。虽然我们专注于检测交叉污染的 rAAV DNA 和重组事件，我们的 Python 脚本可以定制以检测其他序列或事件，例如质粒骨架或来自包装细胞系的 DNA 的反向包装。我们发现 rAAV DNA 提取物的 NGS，称为病毒基因组测序，是一种简单而强大的 rAAV 验证方法。