Abstract
Mutation mapping in mice can be readily accomplished by genome wide segregation analysis of polymorphic DNA markers. In this study, we showed the efficacy of Ion Torrent next generation sequencing for conducting genome-wide scans to map and identify a mutation causing congenital heart disease in a mouse mutant, Bishu, recovered from a mouse mutagenesis screen. The Bishu mutant line generated in a C57BL/6J (B6) background was intercrossed with another inbred strain, C57BL/10J (B10), and the resulting B6/B10 hybrid offspring were intercrossed to generate mutants used for the mapping analysis. For each mutant sample, a panel of 123 B6/B10 polymorphic SNPs distributed throughout the mouse genome was PCR amplified, bar coded, and then pooled to generate a single library used for Ion Torrent sequencing. Sequencing carried out using the 314 chip yielded >600,000 usable reads. These were aligned and mapped using a custom bioinformatics pipeline. Each SNP was sequenced to a depth >500×, allowing accurate automated calling of the B6/B10 genotypes. This analysis mapped the mutation in Bishu to an interval on the proximal region of mouse chromosome 4. This was confirmed by parallel capillary sequencing of the 123 polymorphic SNPs. Further analysis of genes in the map interval identified a splicing mutation in Dnaic1 c.204+1G>A, an intermediate chain dynein, as the disease causing mutation in Bishu. Overall, our experience shows Ion Torrent amplicon sequencing is high throughput and cost effective for conducting genome-wide mapping analysis and is easily scalable for other high volume genotyping analyses.
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Acknowledgments
This study was supported by NIH Grants U01-HL098180 and P30-HL101322. We thank members of the Lo laboratory, Ashok Srinivasan, Mark Kimak and Yang Li for technical support. We thank Dr. Bruce Beutler and Yu Xia for helpful discussions and providing information on the panel of B6/B10 SNP markers ahead of publication.
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The authors declare that they have no competing interests.
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The raw Ion Torrent PGM sequencing data sets supporting the results of this article are available for download from the following link: http://apps.devbio.pitt.edu/Genome/RawData_PGM.zip.
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335_2013_9494_MOESM1_ESM.mov
Videomicroscopy show ciliary motion in the tracheal and ependymal tissue of wildtype (+/+) vs. homozygous Dnaic1 mutant (m/m) mice. The wildtype airway and ependymal tissue showed rapid synchronous ciliary motion, while in the homozygous mutant, the cilia were either immotile or exhibited only slow dyskinetic motion. Beads added to the medium show strong flow was generated in the wildtype, but not in the homozygous mutant trachea and ependymal epithelia
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Damerla, R.R., Chatterjee, B., Li, Y. et al. Ion Torrent sequencing for conducting genome-wide scans for mutation mapping analysis. Mamm Genome 25, 120–128 (2014). https://doi.org/10.1007/s00335-013-9494-7
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DOI: https://doi.org/10.1007/s00335-013-9494-7