Dataset Information

High Density Rice Array (HDRA6.4) developed by the McCouch-Rice Lab at Cornell University

ABSTRACT: A High Density Rice Array (HDRA) was developed as an Affymetrix Custom GeneChip Array by the McCouch Rice Lab at Cornell University. The HDRA assays 700,000 SNPs, or approximately one SNP every 0.54 Kb across the rice genome (genome size = 380 Mb). It was designed to capture most of the haplotype variation observed in a discovery panel consisting of 16M SNPs (generated by sequencing 125 rice genomes at ~7X genome coverage) and to maximize the inclusion of non-synonymous SNPs. Six probes per SNP target were designed as 3 A-allele and 3 B-allele probes at offsets from center ranging from -6 to +6. A small fraction of SNPs have only 4 probes (2-A, 2-B). For all SNPs, the “A” allele is the reference allele (Os-Nipponbare-Reference-IRGSP-1.0 assembly). Additionally, we designed 23,656 x 25-bp probes complimentary to invariant regions of the genome that were used to normalize systematic differences between samples. An estimated 45% of HDRA SNPs map within genes, hitting all 39,045 unique, non-TE rice gene models (MSUv7 rice genome annotation, GFF3 file, Feb. 7, 2012, http://rice.plantbiology.msu.edu/), while 55% of SNPs map to intergenic regions. Non-synonymous are found in 91% of unique, non-TE gene models, and 57% of genic SNPs are distributed within exons, 36% within introns, 5% within 5’ UTRs and 2% within 3’ UTRs. Of the intergenic SNPs, 40% are located in putative regulatory regions within 2 Kb of a transcriptional start site.

ORGANISM(S): Oryza sativa

PROVIDER: GSE71553 | GEO | 2016/02/25

SECONDARY ACCESSION(S): PRJNA291537

REPOSITORIES: GEO

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Project description:In the study of gene expression regulation in plants, it is often desirable to distinguish transcript pools derived from different alleles present in the same organism. We report here an oligonucleotide tiling microarray designed to specifically target 518 single nucleotide polymorphisms (SNPs) between the two sequenced rice (Oryza sativa) subspecies indica and japonica. The tiling array includes all pairs of 25-mer allele-specific probes interrogating each SNP by placing the polymorphic site at all 25 possible positions within the probe. Hybridization of the tiling array to a titration series in which the japonica- and indica-derived cDNA templates are mixed with altering proportions and development of a novel bioinformatic methodology allowed us to screen for diagnostic probe pairs for each SNP. Our result indicates that 284 (55%) SNPs have at least one diagnostic probe pair suitable for distinguishing and quantifying the relative abundance of allele-specific transcripts. As a proof-of-concept, we analyzed allele-specific expression in reciprocal indica × japonica F1 hybrids and detected imbalanced expression at 95 (33%) and 71 (25%) SNPs, respectively. Comparison results from RNA-sequencing and allele-specific real-time PCR experiment validates the sensitivity and reliability of the tiling array method. Together, our results demonstrate the advantages of the tiling array method in interrogating large numbers of SNPs and for the reliability of the experimental and analytical techniques used for quantifying allele-specific gene expression. For each selected SNP, 25 pairs of 25-mer oligonucleotide probes were designed with one match perfectly to the indica allele and the other the japonica allele within each pair. Among the 25 probe pairs, the polymorphic site was placed at a different position such that all possible 25-mer allele-specific probes spanning the polymorphic site were included. The resultant 25,900 probes were synthesized in triplicate in a single microarray produced on the Maskless Array Synthesizer platform as previously described (Li et al., 2005; 2006). Poly(A+) RNA from japonica, indica and their reciprocal hybrids was reverse transcribed using an oligo(dT)18 primer, during which amino-allyl-modified dUTP (aa-dUTP) was incorporated. The aa-dUTP decorated cDNA was fluorescent labeled by conjugating the monofunctional Cy3 dye (GE Healthcare) to the amino-allyl functional groups in the cDNA. Dye-labeled target was quantified using a spectrophotometer and two μg used for hybridization to each array. In the titration series experiment, the dye-labeled targets from japonica and indica were mixed in known proportions before hybridization.

Project description:Transcript abundance results from the balance between transcription and mRNA decay, and varies pervasively in humans. We have examined the effect of DNA variation on mRNA half-life differences by conducting a genome-wide survey of mRNA stability in seven human HapMap lymphoblastoid cell lines (LCLs). We determined the mRNA half-life for each gene from the ratio of 4-thio-uridine (4sU)-labeled nascent RNAs to total RNAs. 5,145 (46%) of 11,132 analyzed genes showed inter-individual mRNA half-life differences at a false discovery rate, FDR<0.05. As previously reported, we found transcription to be the main factor influencing transcript abundance. Although mRNA half-life explained only ~6% of transcript abundance on average, it explained ~16% for the subset of genes (~10%) showing inter-individual mRNA half-life differences (P<0.001). We confirmed previously reported correlations of mRNA half-life with transcript length, 3M-bM-^@M-^Y-UTR length, and number of exon-junctions per kb of transcript. The number of miRNA targets in 3M-bM-^@M-^Y-UTRs was negatively correlated with half-life (P=2.2M-CM-^W10-16), a new observation that is consistent with the role of miRNA in inducing mRNA degradation. Notably, coding GC and GC3 content showed positive correlations with mRNA half-life in genes with inter-individual mRNA half-life differences, implying a role of mRNA stability in shaping synonymous codon usage bias. Consistently, G or C alleles of coding SNPs were found associated with longer mRNA half-life (P=0.021). As expected, we also found that nonsense SNPs were associated with shorter mRNA half-life (P=0.009). Our results strongly suggest that inter-individual mRNA stability differences are widespread and affected by DNA sequence and composition variation. A total of 7 HapMap LCLs were used to measure mRNA half-life. Total RNAs and the 4sU-labeled-newly synthesized RNAs (nascent RNAs) were isolated from the same cell culture and were assayed simultaneously with human Exon array. For 3 LCLs, we included 3 biological replicates (i.e., independent cell cultures) and for 1 LCL we also included technical duplicates. mRNA half-life was calculated from the ratio of nascent RNAs/total RNAs. We used ANOVA to test inter-individual difference of mRNA half-life between 3 subjects who have biological replicates and technical duplicates. We examined the Spearman rank correlation of mRNA half-life with a number of gene features, including transcript length, intron length, 5'-UTR length and folding energy, 3'-UTR length and folding energy, microRNA target sites, GC and GC3 contents, etc. We also performed linear regression to test the effects of specific type of sequence variants (nonsense SNPs, SNPs within miRNA target sites, and coding synonymous and nonsynonymous SNPs) on mRNA half-life across 3 subjects that have whole genome sequencing data available (1000 genome project June 2011 release).

Dataset Information

High Density Rice Array (HDRA6.4) developed by the McCouch-Rice Lab at Cornell University

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