ASIS-Seq: Transgene Insertion Site Mapping by Nanopore Adaptive Sampling

Overview

Journal Methods Mol Biol

Specialty Molecular Biology

Date 2023 Mar 30

PMID 36995666

Authors

Charles Yu

Roger Caothien

Anna Pham

Lucinda Tam

Tuija Alcantar

Natasha Bacarro

Juan Reyes Jr

Marques Jackson

Brian Nakao

Merone Roose-Girma

Affiliations

Soon will be listed here.

Abstract

Generation of transgenic mice by direct microinjection of foreign DNA into fertilized ova has become a routine technique in biomedical research. It remains an essential tool for studying gene expression, developmental biology, genetic disease models, and their therapies. However, the random integration of foreign DNA into the host genome that is inherent to this technology can lead to confounding effects associated with insertional mutagenesis and transgene silencing. Locations of most transgenic lines remain unknown because the methods are often burdensome (Nicholls et al., G3: Genes Genomes Genetics 9:1481-1486, 2019) or have limitations (Goodwin et al., Genome Research 29:494-505, 2019). Here, we present a method that we call Adaptive Sampling Insertion Site Sequencing (ASIS-Seq) to locate transgene integration sites using targeted sequencing on Oxford Nanopore Technologies' (ONT) sequencers. ASIS-Seq requires only about 3 ug of genomic DNA, 3 hours of hands-on sample preparation time, and 3 days of sequencing time to locate transgenes in a host genome.

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