TY - JOUR

T1 - MYSTIC-seq

T2 - plasmid-based hybridization capture for mapping transgene integration sites in transgenic mice

AU - Battulin, Nariman

AU - Savoskin, Maxim

AU - Maltseva, Ekaterina

AU - Smirnov, Alexander

AU - Gridina, Maria

N1 - Battulin, N., Savoskin, M., Maltseva, E. et al. MYSTIC-seq: plasmid-based hybridization capture for mapping transgene integration sites in transgenic mice. Transgenic Res 35, 43 (2026). https://doi.org/10.1007/s11248-026-00524-6 Funding Ministry of Education and Science of the Russian Federation, 075-15-2024-539

PY - 2026/10/27

Y1 - 2026/10/27

N2 - Characterizing the genomic integration site of a transgene is essential for interpreting phenotypes in transgenic animal lines and for designing reliable PCR-based genotyping assays that distinguish hemizygous, homozygous, and wild-type animals. Existing methods for integration-site mapping including inverse PCR, TAIL-PCR, Targeted Locus Amplification (TLA), and Cas9-mediated long-read sequencing vary in cost, throughput, infrastructure requirements, and accessibility. Here we describe MYSTIC-seq (Mapping Your Secret Transgene Integration Coordinates), a targeted short-read sequencing approach in which the capture probe library is generated directly from the plasmid used to produce the transgenic animals. This eliminates the need to design and synthesize individual capture oligonucleotides and makes the method immediately adaptable to any construct for which the plasmid is available. Genomic DNA libraries are enriched by hybridization with biotinylated plasmid-derived probes, captured on streptavidin magnetic beads, and sequenced on a standard Illumina platform at modest depth (~ 1 million reads per sample). Integration sites are identified by local enrichment of genomic coverage adjacent to the insertion together with discordant and split reads spanning transgene-genome junctions. We demonstrate the method on three transgenic mouse lines generated by pronuclear microinjection. In the ACE5 line, both transgene-genome junctions were mapped, revealing a ~54 kb deletion at the integration site; the recovered coordinates enabled a PCR genotyping assay that distinguished zygosity and allowed the homozygous line to be established without test crosses. In the MT1 and APP lines, one of two junctions was recovered, sufficient for genotyping, and analysis of the MT1 line detected incorporation of vector backbone sequence into the concatemer, a known artifact of pronuclear microinjection. We discuss the limitations of the approach, including incomplete junction recovery for complex concatemeric loci, masking of junctions within Cot-1-suppressed repetitive regions, and the inability to detect ectopic non-reference DNA incorporated into the concatemer. MYSTIC-seq provides a low-cost, accessible, and readily adaptable solution for the most common practical need in transgenic animal facilities: rapid identification of integration-site coordinates for genotyping and colony management.

AB - Characterizing the genomic integration site of a transgene is essential for interpreting phenotypes in transgenic animal lines and for designing reliable PCR-based genotyping assays that distinguish hemizygous, homozygous, and wild-type animals. Existing methods for integration-site mapping including inverse PCR, TAIL-PCR, Targeted Locus Amplification (TLA), and Cas9-mediated long-read sequencing vary in cost, throughput, infrastructure requirements, and accessibility. Here we describe MYSTIC-seq (Mapping Your Secret Transgene Integration Coordinates), a targeted short-read sequencing approach in which the capture probe library is generated directly from the plasmid used to produce the transgenic animals. This eliminates the need to design and synthesize individual capture oligonucleotides and makes the method immediately adaptable to any construct for which the plasmid is available. Genomic DNA libraries are enriched by hybridization with biotinylated plasmid-derived probes, captured on streptavidin magnetic beads, and sequenced on a standard Illumina platform at modest depth (~ 1 million reads per sample). Integration sites are identified by local enrichment of genomic coverage adjacent to the insertion together with discordant and split reads spanning transgene-genome junctions. We demonstrate the method on three transgenic mouse lines generated by pronuclear microinjection. In the ACE5 line, both transgene-genome junctions were mapped, revealing a ~54 kb deletion at the integration site; the recovered coordinates enabled a PCR genotyping assay that distinguished zygosity and allowed the homozygous line to be established without test crosses. In the MT1 and APP lines, one of two junctions was recovered, sufficient for genotyping, and analysis of the MT1 line detected incorporation of vector backbone sequence into the concatemer, a known artifact of pronuclear microinjection. We discuss the limitations of the approach, including incomplete junction recovery for complex concatemeric loci, masking of junctions within Cot-1-suppressed repetitive regions, and the inability to detect ectopic non-reference DNA incorporated into the concatemer. MYSTIC-seq provides a low-cost, accessible, and readily adaptable solution for the most common practical need in transgenic animal facilities: rapid identification of integration-site coordinates for genotyping and colony management.

KW - Integration-site mapping

KW - Targeted sequencing

KW - Transgene integration

KW - Transgenic mice

KW - Интеграция трансгена

KW - Трансгенные мыши

KW - аргетное секвенирование

KW - Картирование сайтов интеграции

UR - https://www.mendeley.com/catalogue/47d714c4-ddfc-3d36-bf34-dac2572fe8ec/

UR - https://www.scopus.com/pages/publications/105054724241

U2 - 10.1007/s11248-026-00524-6

DO - 10.1007/s11248-026-00524-6

M3 - Article

VL - 35

JO - Transgenic Research

JF - Transgenic Research

SN - 0962-8819

IS - 1

M1 - 43

ER -