Ion Torrent sequencing for conducting genome-wide scans for mutation mapping analysis

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 Dnaic1c.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.

Global genetic analysis in mice unveils central role for cilia in congenital heart disease

“…In mutant lines with multiple homozygous mutations on the same chromosome linked to the CHD phenotype, the pathogenic mutation was identified with segregation analysis of additional mutants, and/or based on phenotype comparison to existing knockout/mutant alleles. For five lines (b2b002/b2b012/b2b284/b2b1519/b2b1146), the mutation was confirmed with mapping analyses 13 .…”

Activation and function of murine primary microglia in the absence of the prion protein

“…The C57BL/6J and C57BL/10SnJ mice have been separately inbred since 1947 (JAX notes -http://jaxmice.jax.org/strain/000665. html), and expression of certain genes, single nucleotide polymorphisms, as well as functional properties of a variety of organs and systems have, indeed, been shown to differ between mice of either strain (Steward et al, 1999;Shuttleworth and Connor, 2001;Turk et al, 2004;Deacon et al, 2007;Xia et al, 2010;Wiltshire et al, 2011;Roche et al, 2012;Damerla et al, 2014). Undoubtedly, the interplay of both Prnp and Sirpa genes and their products deserves further attention, to unravel both their genetic and functional interactions, which may affect not only immune cells but functional properties attributed to PrP C in other organs and systems.…”

Preimplantation genetic testing for aneuploidy: what technology should you use and what are the differences?

“…These are the MiSeq from Illumina and the Personal Genome Machine (PGM) from Thermo-Fisher Scientific [37,38,[51][52][53]. Following DNA amplification, approximately 50 ng of each DNA sample is enzymatically digested into millions of fragments and pooled for library preparation.…”