Detection of a complex chromosomal rearrangement in a novel mouse mutant by optical genome mapping
Description
The purpose of this study was to identify the genomic rearrangements present in a transgenic mouse line (Line 781) that expresses a lacZ reporter gene cassette. Mice hemizygous for the transgene have a mutant phenotype (smaller body size and paws, and craniofacial aberrations), whereas their non-transgenic littermates are phenotypically normal. The transgene inserted near or at a reciprocal translocation (RT) between chromosomes 1 and 2 as determined by fluorescent in situ hybridization (FISH) analysis. Optical genome mapping (OGM) was performed to better identify the genomic rearrangements present in the mutant mouse genome. OGM was performed with ultra-high molecular weight genomic DNA isolated from a mutant female mouse, with an average length of 246.092 kbp. The DNA was labeled with DL-green fluorophores at CTTAAG sites with DLE-1 methyltransferase. Because the target site is palindromic, both DNA strands get labeled. The label density averaged 15.017 labels per 100 kb. The labeled DNA was analyzed on the Bionano Saphyr system to measure the distance between labels (tags) for each single, extremely long strand of genomic DNA (‘molecule’). The resulting data was entered into the de novo assembly pipeline to group the molecules by label pattern similarity in order to create maps, which then were used to construct the mutant genome. The rare variant analysis pipeline was used to compare the assembly against a reference genome (GRCm38.p6). Areas of discrepancy within the mutant genome were identified using the structural variant (SV) caller. It indicated that the RT junction in der2 was separated by extraneous DNA, which happens to be the transgene. Based on target sites for labeling within the transgene sequence, it was estimated that five complete copies of the transgene integrated in a tail-to-head orientation, directly between the breakpoints in der2. The breakpoints in der2 were estimated to be in the vicinity of positions chr2:106,476,853 and chr1:163,466,814. OGM results also estimated the RT junction in der1 occurs near positions chr1:159,566,643 and chr2:108,255,381. Taken together, the data suggest that parts of chromosomes 1 and 2 were deleted during the reorganization of the mutant genome. As well, copy number variant (CNV) analysis was performed for the mutant genome using the Bionano software. The analysis confirmed areas of monosomy within chromosomes 1 and 2, which correspond to gaps of missing sequence in the derivative chromosomes when the SV caller estimated breakpoints are considered in combination. Thus, the RT is unbalanced, with segments of chr1 (3.9 Mbp) and chr2 (1.8 Mbp) missing from the derivative chromosomes. The breakpoints were identified precisely by Sanger sequencing of PCR products that span a junction between chromosomal breakpoints or with the transgene. Thus, it is likely that the phenotype in mutant animals is due to the deletion of a gene or genes within the deleted sequence.
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Steps to reproduce
OGM sample preparation, mapping, and bioinformatics analysis was conducted by the Molecular Genomics core at Texas A&M University (College Station, TX, USA). Ultra-high molecular weight genomic DNA isolated from the liver of a mutant female mouse from Line 781 (Patyal et al., 2020; doi:10.1007/s11064-019-02919-w), with an average length of 246.092 kbp. The DNA was labeled with DL-green fluorophores at CTTAAG sites with DLE-1 methyltransferase. The labeled DNA was analyzed on the Bionano Saphyr system to measure the distance between labels (tags) for each single, extremely long strand of genomic DNA (‘molecule’). The resulting data was entered into the de novo assembly pipeline to group the molecules by label pattern similarity in order to create maps, which then were used to construct the mutant genome. The rare variant analysis pipeline was used to compare the assembly against a reference genome (GRCm38.p6). Areas of discrepancy within the mutant genome were identified using the structural variant (SV) caller. As well, copy number variant (CNV) analysis was performed for the mutant genome using the Bionano software (version 1.6.1). The analysis confirmed areas of monosomy within chromosomes 1 and 2, which correspond to gaps of missing sequence from the derivative chromosomes when the SV caller estimated breakpoints are considered in combination. ZIP files can be extracted, uploaded and viewed using the Bionano Access software. Maps 1344 and 1345 were used to identify the junctions of the unbalanced reciprocal translocation.
Institutions
- University of Arkansas for Medical SciencesArkansas, Little Rock
Categories
Funders
- UAMS Barton Pilot Study