Dataset Information

Genome-wide integration site detection using Cas9 enriched amplification-free long-range sequencing.

ABSTRACT: The gene and cell therapy fields are advancing rapidly, with a potential to treat and cure a wide range of diseases, and lentivirus-based gene transfer agents are the vector of choice for many investigators. Early cases of insertional mutagenesis caused by gammaretroviral vectors highlighted that integration site (IS) analysis was a major safety and quality control checkpoint for lentiviral applications. The methods established to detect lentiviral integrations using next-generation sequencing (NGS) are limited by short read length, inadvertent PCR bias, low yield, or lengthy protocols. Here, we describe a new method to sequence IS using Amplification-free Integration Site sequencing (AFIS-Seq). AFIS-Seq is based on amplification-free, Cas9-mediated enrichment of high-molecular-weight chromosomal DNA suitable for long-range Nanopore MinION sequencing. This accessible and low-cost approach generates long reads enabling IS mapping with high certainty within a single day. We demonstrate proof-of-concept by mapping IS of lentiviral vectors in a variety of cell models and report up to 1600-fold enrichment of the signal. This method can be further extended to sequencing of Cas9-mediated integration of genes and to in vivo analysis of IS. AFIS-Seq uses long-read sequencing to facilitate safety evaluation of preclinical lentiviral vector gene therapies by providing IS analysis with improved confidence.

SUBMITTER: van Haasteren J

PROVIDER: S-EPMC7897500 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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Genome-wide integration site detection using Cas9 enriched amplification-free long-range sequencing.

van Haasteren Joost J Munis Altar M AM Gill Deborah R DR Hyde Stephen C SC

Nucleic acids research 20210201 3

The gene and cell therapy fields are advancing rapidly, with a potential to treat and cure a wide range of diseases, and lentivirus-based gene transfer agents are the vector of choice for many investigators. Early cases of insertional mutagenesis caused by gammaretroviral vectors highlighted that integration site (IS) analysis was a major safety and quality control checkpoint for lentiviral applications. The methods established to detect lentiviral integrations using next-generation sequencing ( ...[more]

PMID: 33290561

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Project description:UnlabelledAntiretroviral therapy (ART) is successful in the suppression of HIV but cannot target and eradicate the latent proviral reservoir. The location of retroviral integration into the human genome is thought to play a role in the clonal expansion of infected cells and HIV persistence. We developed a high-throughput targeted sequence capture assay that uses a pool of HIV-specific probes to enrich Illumina libraries prior to deep sequencing. Using an expanded clonal population of ACH-2 cells, we demonstrate that this sequence capture assay has an extremely low false-positive rate. This assay assessed four cellular models commonly used to study HIV latency and latency-reversing agents: ACH-2 cells, J-Lat cells, the Bcl-2-transduced primary CD4(+)model, and the cultured TCM(central memory) CD4(+)model. HIV integration site characteristics and genes were compared between these cellular models and to previously reported patient data sets. Across these cellular models, there were significant differences in integration site characteristics, including orientation relative to that of the host gene, the proportion of clonally expanded sites, and the proportion located within genic regions and exons. Despite a greater diversity of minority integration sites than expected in ACH-2 cells, their integration site characteristics consistently differed from those of the other models and from the patient samples. Gene ontology analysis of highly represented genes from the patient samples found little overlap with HIV-containing genes from the cell lines. These findings show that integration site differences exist among the commonly used cellular models of HIV latency and in comparison to integration sites found in patient samples.ImportanceDespite the success of ART, currently there is no successful therapy to eradicate integrated proviruses. Cellular models of HIV latency are used to test the efficacy of latency-reversing agents, but it is unclear how well these models reflect HIV integration into the human genome in vivo We have developed a novel probe-based sequence enrichment assay to sequence and analyze integrated HIV. We compared HIV integration site characteristics between four cellular models and to previously described patient data sets. Significant differences were detected in the distribution of HIV integration sites between cellular models of HIV latency and compared to data sets from patient samples. The results from this study have implications for how well these cellular models of HIV infection truly reflect HIV integration in vivo and their applicability in drug discovery for novel latency-reversing agents.

Dataset Information

Genome-wide integration site detection using Cas9 enriched amplification-free long-range sequencing.

Publications

Genome-wide integration site detection using Cas9 enriched amplification-free long-range sequencing.

Similar Datasets

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