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Highly parallel SNP genotyping reveals high-resolution landscape of mono-allelic Ube3a expression associated with locus-wide antisense transcription.

ABSTRACT: We investigated the allele- and strand-specific transcriptional landscape of a megabase-wide genomic region of mouse Ube3a (ubiquitin protein ligase E3A) by means of a highly parallel SNP genotyping platform. We have successfully identified maternal-specific expression of Ube3a and its antisense counterpart (Ube3a-ATS) in brain, but not in liver. Because of the use of inter-subspecies hybrid mice, this megabase-wide analysis provided high-resolution picture of the transcriptional patterns of this region. First, we showed that brain-specific maternal expression of Ube3a is restricted to the second half part of the locus, but is absent from the first half part. Balance of allelic expression is altered in the middle of the locus. Second, we showed that expression of the brain-specific Ube3a-ATS appeared to be terminated in the region upstream to the Ube3a transcription start site. The present study highlights the importance of locus-wide competition between sense and antisense transcripts.

SUBMITTER: Numata K

PROVIDER: S-EPMC3074135 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

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Highly parallel SNP genotyping reveals high-resolution landscape of mono-allelic Ube3a expression associated with locus-wide antisense transcription.

Numata Koji K Kohama Chihiro C Abe Kuniya K Kiyosawa Hidenori H

Nucleic acids research 20101203 7

We investigated the allele- and strand-specific transcriptional landscape of a megabase-wide genomic region of mouse Ube3a (ubiquitin protein ligase E3A) by means of a highly parallel SNP genotyping platform. We have successfully identified maternal-specific expression of Ube3a and its antisense counterpart (Ube3a-ATS) in brain, but not in liver. Because of the use of inter-subspecies hybrid mice, this megabase-wide analysis provided high-resolution picture of the transcriptional patterns of thi ...[more]

PMID: 21131283

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Project description:BackgroundTogether single nucleotide substitutions and small insertion/deletion variants are the most common form of sequence variation in the human gene pool.High-resolution SNP profile and/or haplotype analyses enable the identification of modest-risk susceptibility genes to common diseases, genes that may modulate responses to pharmaceutical agents, and SNPs that can affect either their expression or function. In addition, sensitive techniques for germline or somatic mutation detection are important tools for characterizing sequence variations in genes responsible for tumor predisposition. Cost-effective methods are highly desirable. Many of the recently developed high-throughput technologies are geared toward industrial scale genetic studies and arguably do not provide useful solutions for small laboratory investigator-initiated projects. Recently, the use of new fluorescent dyes allowed the high-resolution analysis of DNA melting curves (HRM).ResultsHere, we compared the capacity of HRM, applicable to both genotyping and mutation scanning, to detect genetic variations in the tumor suppressor gene TP53 with that of mutation screening by full resequencing. We also assessed the performance of a variety of available HRM-based genotyping assays by genotyping 30 TP53 SNPs. We describe a series of solutions to handle the difficulties that may arise in large-scale application of HRM to mutation screening and genotyping at the TP53 locus. In particular, we developed specific HRM assays that render possible genotyping of 2 or more, sometimes closely spaced, polymorphisms within the same amplicon. We also show that simultaneous genotyping of 2 SNPs from 2 different amplicons using a multiplex PCR reaction is feasible; the data can be analyzed in a single HRM run, potentially improving the efficiency of HRM genotyping workflows.ConclusionThe HRM technique showed high sensitivity and specificity (1.0, and 0.8, respectively, for amplicons of <400 bp) for mutation screening and provided useful genotyping assays as assessed by comparing the results with those obtained with Sanger sequencing. Thus, HRM is particularly suitable for either performing mutation scanning of a large number of samples, even in the situation where the amplicon(s) of interest harbor a common variant that may disturb the analysis, or in a context where gathering common SNP genotypes is of interest.

Project description:The genome-wide discovery and high-throughput genotyping of SNPs in chickpea natural germplasm lines is indispensable to extrapolate their natural allelic diversity, domestication, and linkage disequilibrium (LD) patterns leading to the genetic enhancement of this vital legume crop. We discovered 44,844 high-quality SNPs by sequencing of 93 diverse cultivated desi, kabuli, and wild chickpea accessions using reference genome- and de novo-based GBS (genotyping-by-sequencing) assays that were physically mapped across eight chromosomes of desi and kabuli. Of these, 22,542 SNPs were structurally annotated in different coding and non-coding sequence components of genes. Genes with 3296 non-synonymous and 269 regulatory SNPs could functionally differentiate accessions based on their contrasting agronomic traits. A high experimental validation success rate (92%) and reproducibility (100%) along with strong sensitivity (93-96%) and specificity (99%) of GBS-based SNPs was observed. This infers the robustness of GBS as a high-throughput assay for rapid large-scale mining and genotyping of genome-wide SNPs in chickpea with sub-optimal use of resources. With 23,798 genome-wide SNPs, a relatively high intra-specific polymorphic potential (49.5%) and broader molecular diversity (13-89%)/functional allelic diversity (18-77%) was apparent among 93 chickpea accessions, suggesting their tremendous applicability in rapid selection of desirable diverse accessions/inter-specific hybrids in chickpea crossbred varietal improvement program. The genome-wide SNPs revealed complex admixed domestication pattern, extensive LD estimates (0.54-0.68) and extended LD decay (400-500 kb) in a structured population inclusive of 93 accessions. These findings reflect the utility of our identified SNPs for subsequent genome-wide association study (GWAS) and selective sweep-based domestication trait dissection analysis to identify potential genomic loci (gene-associated targets) specifically regulating important complex quantitative agronomic traits in chickpea. The numerous informative genome-wide SNPs, natural allelic diversity-led domestication pattern, and LD-based information generated in our study have got multidimensional applicability with respect to chickpea genomics-assisted breeding.

Dataset Information

Highly parallel SNP genotyping reveals high-resolution landscape of mono-allelic Ube3a expression associated with locus-wide antisense transcription.

Publications

Highly parallel SNP genotyping reveals high-resolution landscape of mono-allelic Ube3a expression associated with locus-wide antisense transcription.

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