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Exon capture and bulk segregant analysis: rapid discovery of causative mutations using high-throughput sequencing.

del Viso F, Bhattacharya D, Kong Y, Gilchrist MJ, Khokha MK - BMC Genomics (2012)

Bottom Line: We demonstrate that bulk segregant analysis coupled with exon capture sequencing is not only able to identify causative mutations but can also generate linkage information, facilitate the assembly of scaffolds, identify misassembles, and discover thousands of SNPs for fine mapping.Exon capture sequencing and bulk segregant analysis is a rapid, inexpensive method to clone mutants identified in forward genetic screens.With sufficient meioses, this method can be generalized to any model system with a genome assembly, polished or unpolished, and in the latter case, it also provides many critical genomic resources.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pediatrics and Genetics, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA.

ABSTRACT

Background: Exome sequencing has transformed human genetic analysis and may do the same for other vertebrate model systems. However, a major challenge is sifting through the large number of sequence variants to identify the causative mutation for a given phenotype. In models like Xenopus tropicalis, an incomplete and occasionally incorrect genome assembly compounds this problem. To facilitate cloning of X. tropicalis mutants identified in forward genetic screens, we sought to combine bulk segregant analysis and exome sequencing into a single step.

Results: Here we report the first use of exon capture sequencing to identify mutations in a non-mammalian, vertebrate model. We demonstrate that bulk segregant analysis coupled with exon capture sequencing is not only able to identify causative mutations but can also generate linkage information, facilitate the assembly of scaffolds, identify misassembles, and discover thousands of SNPs for fine mapping.

Conclusion: Exon capture sequencing and bulk segregant analysis is a rapid, inexpensive method to clone mutants identified in forward genetic screens. With sufficient meioses, this method can be generalized to any model system with a genome assembly, polished or unpolished, and in the latter case, it also provides many critical genomic resources.

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ruby and grinch mutant phenotypes. ruby (a) and grinch (b) mutant phenotypes start with pericardial edema at stage (st.) 39. At st. 45, the edema worsens and is lethal. Wildtype (WT) embryos from the same cross are shown for comparison. All embryos are lateral views with dorsal to the top and anterior to the left.
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Figure 1: ruby and grinch mutant phenotypes. ruby (a) and grinch (b) mutant phenotypes start with pericardial edema at stage (st.) 39. At st. 45, the edema worsens and is lethal. Wildtype (WT) embryos from the same cross are shown for comparison. All embryos are lateral views with dorsal to the top and anterior to the left.

Mentions: In a forward genetic screen in X. tropicalis, we identified two mutants, ruby and grinch that follow simple Mendelian inheritance, show similar phenotypes, but fall into different complementation groups (Figure 1). Both mutants appear wildtype until stage 38–39 when edema starts to appear around the heart, steadily worsens, and finally causes death by stage 46–48 (Figure 1a,b). The etiology of the edema is unclear and could be due to cardiac, lymphatic or renal defects [29-34]. To determine the causative genes, we developed a HTS approach combining exon capture with BSA. First, we developed an exon capture array containing coding exons identified in the published X. tropicalis v4.1 genome [35]. Because approximately 5-10% of the genome is missing from this assembly [18,35], we also identified coding exons from available EST clusters and full-length mRNA sequences to augment the array.


Exon capture and bulk segregant analysis: rapid discovery of causative mutations using high-throughput sequencing.

del Viso F, Bhattacharya D, Kong Y, Gilchrist MJ, Khokha MK - BMC Genomics (2012)

ruby and grinch mutant phenotypes. ruby (a) and grinch (b) mutant phenotypes start with pericardial edema at stage (st.) 39. At st. 45, the edema worsens and is lethal. Wildtype (WT) embryos from the same cross are shown for comparison. All embryos are lateral views with dorsal to the top and anterior to the left.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3526394&req=5

Figure 1: ruby and grinch mutant phenotypes. ruby (a) and grinch (b) mutant phenotypes start with pericardial edema at stage (st.) 39. At st. 45, the edema worsens and is lethal. Wildtype (WT) embryos from the same cross are shown for comparison. All embryos are lateral views with dorsal to the top and anterior to the left.
Mentions: In a forward genetic screen in X. tropicalis, we identified two mutants, ruby and grinch that follow simple Mendelian inheritance, show similar phenotypes, but fall into different complementation groups (Figure 1). Both mutants appear wildtype until stage 38–39 when edema starts to appear around the heart, steadily worsens, and finally causes death by stage 46–48 (Figure 1a,b). The etiology of the edema is unclear and could be due to cardiac, lymphatic or renal defects [29-34]. To determine the causative genes, we developed a HTS approach combining exon capture with BSA. First, we developed an exon capture array containing coding exons identified in the published X. tropicalis v4.1 genome [35]. Because approximately 5-10% of the genome is missing from this assembly [18,35], we also identified coding exons from available EST clusters and full-length mRNA sequences to augment the array.

Bottom Line: We demonstrate that bulk segregant analysis coupled with exon capture sequencing is not only able to identify causative mutations but can also generate linkage information, facilitate the assembly of scaffolds, identify misassembles, and discover thousands of SNPs for fine mapping.Exon capture sequencing and bulk segregant analysis is a rapid, inexpensive method to clone mutants identified in forward genetic screens.With sufficient meioses, this method can be generalized to any model system with a genome assembly, polished or unpolished, and in the latter case, it also provides many critical genomic resources.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pediatrics and Genetics, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA.

ABSTRACT

Background: Exome sequencing has transformed human genetic analysis and may do the same for other vertebrate model systems. However, a major challenge is sifting through the large number of sequence variants to identify the causative mutation for a given phenotype. In models like Xenopus tropicalis, an incomplete and occasionally incorrect genome assembly compounds this problem. To facilitate cloning of X. tropicalis mutants identified in forward genetic screens, we sought to combine bulk segregant analysis and exome sequencing into a single step.

Results: Here we report the first use of exon capture sequencing to identify mutations in a non-mammalian, vertebrate model. We demonstrate that bulk segregant analysis coupled with exon capture sequencing is not only able to identify causative mutations but can also generate linkage information, facilitate the assembly of scaffolds, identify misassembles, and discover thousands of SNPs for fine mapping.

Conclusion: Exon capture sequencing and bulk segregant analysis is a rapid, inexpensive method to clone mutants identified in forward genetic screens. With sufficient meioses, this method can be generalized to any model system with a genome assembly, polished or unpolished, and in the latter case, it also provides many critical genomic resources.

Show MeSH
Related in: MedlinePlus