del Viso F et al. (2012), Exon capture and bulk segregant analysis: rapid...

XB-ART-46277

BMC Genomics 2012 Nov 21;13:649. doi: 10.1186/1471-2164-13-649.

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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 .

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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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Species referenced: Xenopus tropicalis
Genes referenced: atp1a1 c3h2orf68 ccdc40 cdh16 hnf1a hnf1b il1b maf pax2 pax8 psd4 rgn sftpb slc5a9 slc7a8 tmem150a usp39
GO keywords: ciliary basal body organization [+]
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???displayArticle.disOnts??? primary ciliary dyskinesia

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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.
	Figure 2. BSA and Exon Capture HTS. (a) Schematic of Exon Capture HTS with BSA. Two pools of embryos are sequenced, one mutant and one WT from a mapcross. The WT pool is sequenced and SNPs identified based on allele counts where the major allele fraction (MAF) is approximately 50%. These positions are examined in the mutant pools for LOH. (b,d) Graph of homozygosity ratio to scaffold number using genome v4.1 for ruby (b) and grinch (d). (c,e) same as b,d but version 7.1 of the genome. In (b-e), scaffolds or scaffold intervals with <20 SNPs have been excluded. The peaks with the highest homozygosity ratios are labeled.
	Figure 3. Mapping of ruby mutation. (a) Mapping interval derived from exon capture and BSA. The number of recombinants for each RFLP marker, the genetic distance and the location of the marker in v4.1 and v7.1 genome assemblies are shown. The 220 kb interval contains 7 genes: tmem150a, c2orf68, usp39, sftpb, pax8, psd4 and a portion of il1b. Genes in red have mutations causing amino acid or splice site changes based on the exon capture data. (b) Genomic, cDNA and protein sequences of WT and mutant pax8. The mutation of two contiguous nucleotides (nt) in intron 2 (arrowhead) shifts the acceptor splice site 1 nt, causing the inclusion of an extra G in the transcript (cDNA) (arrowhead). The protein sequence shows a change in frame and shortly after a premature STOP codon (*) abrogating the entire paired box domain. (c) Pax2 expression in wt and mutant ruby embryos by Whole Mount in situ hybridization showing clear impairment of pronephric development (arrowhead). (d) Pax8 morpholino phenocopies ruby phenotype. Pax8 morpholino was injected at one or two-cell stage (single cell injected). Embryos were fixed at st. 36–37 and pax2 expression was used to assess pronephric development. The embryo shown for two-cell stage injection was injected on the right side (based on fluorescent dye tracer [not shown]). Arrowheads indicate abnormal pronephros. Embryos in (c,d) are lateral views with dorsal to the top and anterior to the left (in left columns) or right (in right columns).
	Figure 4. Mapping the grinch mutation. (a) Local genome assembly of the grinch locus using exon capture/BSA. The blue region marks the 10 cM gap in the meiotic map. The number of recombinants for each marker, calculated genetic distance and the location of the markers in v7.1 and v4.1 genome assemblies are shown. The 400 kb interval contains 2 genes: ccdc40 on the ends of scaffold 148 and 304 (v4.1) and ribosomal protein L38. (b) Schematic diagram of the protein sequences of the 21 mutant clones. Red regions indicate deletions, green box indicates insertions, and dashed lines indicate frameshifts. Number of bp inserted or deleted and translated amino acid (a.a.) length for each clone is indicated. See Additional file 1: Figure S5 for complete mutant protein and cDNA sequence. (c) Cilia-driven epidermal flow study using red microbeads. Arrows follow a particular bead’s trajectory through time. In left column, WT embryo shows equal flow on both the left and right sides. In middle column, grinch mutants show no flow on either side. In right column, mutants injected with WT ccdc40 mRNA and GFP tracer (insert) show rescue of flow on injected side. See Additional files 1– 4. Embryos in (c) are dorsal views with anterior to the right.
	Supplementary Figure 1: psd4 expression by WMISH at stages before pronephros are functional (st. 27) and when they are starting to function (st. 38).
	Supplementary Figure 2: Expression of pronephric markers in wt and ruby mutant embryos. Embryos from a heterozygous carrier cross were fixed at 36-37 stage and the expression of pronephric markers: pax8 (paired box 8), pax2 (paired box 2), atp1a1 (ATPase, Na+/K+ transporting, alpha 1 polypeptide) , slc7a8 (solute carrier family 7 (amino acid transporter light chain, L system), member 8), slc5a9 (solute carrier family 5 (sodium/glucose cotransporter), member 9), smp-30 (senescence marker protein-30), hnf1-b (HNF1 homeobox b) and cdh16 (cadherin 16), were studied by WMISH. Embryos were genotyped to confirm the mutation after imaging. Arrowheads indicate abnormal pronephros.
	Supplementary Figure 3: Phenocopy of ruby mutant by pax8 MO injection. Pax8 morpholino was injected at one (a) or two cell stage (single cell injected) (b). Embryos were fixed at st 36-37 and pronephric development was assessed by pax2 expression. Abnormalities in pronephros development were classified: No phenotype; pronephros similar to uninjected controls (UIC); Type I: disrupted and abnormal pronephros similar to ruby mutants on both sides of the embryos. Type II: abnormal pronephros on one side and moderate abnormal pronephros on the other side; Type III: disrupted and abnormal pronephros on the injected side and moderate abnormal phenotype on the uninjected side. Type IV: disrupted and abnormal on the injected side and normal pronephros on the uninjected side. Images of Pax2 WMISH showing each type of phenotype are shown. For Type III and IV, embryos shown were injected on the right side. Arrowheads indicate disrupted and abnormal pronephros.
	Supplementary Figure 4: SEM and TEM of VITT and grinch embryos. (a-f) SEM; (g,h) TEM; (a-c) WT embryos; (d-f) grinch mutant embryos. (f) grinch mutants have fewer, shorter and abnormal cilia per cell. (g) VVT cilia showing the 9+2 microtubule structure. (h) mutant cilia showing a displaced outer ring microtubule doublet (arrow). Embryos in (a,d) are lateral view with dorsal on top and anterior to the right.
	Supplementary Figure 5: (a) Protein sequence alignment of the 21 grinch mutant clones. (b) cDNA sequence of the 21 mutant clones. Heterozygosity at the mutant locus (red arrows).

References [+] :

Abu-Daya, Absence of heartbeat in the Xenopus tropicalis mutation muzak is caused by a nonsense mutation in cardiac myosin myh6. 2009, Pubmed, Xenbase