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Analysis of the transgene insertion pattern in a transgenic mouse strain using long-read sequencing
Osamu Suzuki1, Minako Koura1, Kozue Uchio-Yamada1
1Laboratory of Animal Models for Human Diseases, National Institutes of Biomedical Innovation, Health and Nutrition,7-6-8 Saito-Asagi, Ibaraki, Ibaraki, Osaka 568-0085, Japan.
Experimental Animals
|February 14, 2020
Summary
Long-read sequencing accurately analyzes transgene insertion patterns, including copy number and genomic alterations. This method overcomes previous read-length limitations for comprehensive transgenic animal studies.
Area of Science:
- Genomics
- Molecular Biology
- Transgenic Technology
Background:
- Transgene insertion patterns (copy number, orientation, position) critically influence transgene function in transgenic animals.
- Previous genomic walking strategies had limitations in determining transgene copy number and detecting large genomic modifications due to short read lengths.
Purpose of the Study:
- To overcome read-length limitations in analyzing transgene insertion patterns.
- To characterize transgene insertion site, copy number, and associated genomic modifications using long-read sequencing.
Main Methods:
- Utilized a long-read sequencer (MinION, Oxford Nanopore Technologies) for genomic DNA analysis from a transgenic mouse strain.
- Employed local BLAST and Ensembl's BLAST searches for sequence analysis and genome mapping.
- Performed nucleotide dot plot analysis to determine transgene arrangement.
Main Results:
- Generated over 922,000 long reads, identifying a 21,457-bp read containing the transgene.
- Confirmed tandem concatemer insertion of the transgene with an intact construct and a partial fragment.
- Discovered a 9,388-bp deletion at the Sgcd gene intron insertion site, indicating large genomic alterations during insertion.
Conclusions:
- Long-read sequencing is a powerful tool for comprehensive analysis of transgene insertion patterns.
- This technology enables accurate determination of transgene copy number and detection of associated genomic rearrangements.
- The findings highlight the utility of long-read sequencing for characterizing complex transgene integration events and associated mutations.
