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:Background: Genome-wide detection of single feature polymorphisms (SFP) in swine using transcriptome profiling of day 25 placental RNA by contrasting probe intensities from either Meishan or an occidental composite breed with Affymetrix porcine microarrays is presented. A linear mixed model analysis was used to identify significant breed-by-probe interactions. Results: Gene specific linear mixed models were fit to each of the log2 transformed probe intensities on these arrays, using fixed effects for breed, probe, breed-by-probe interaction, and a random effect for array. After surveying the day 25 placental transcriptome, 789 probes with a q-value ≤ 0.05 and |fold change| ≥ 2 for the breed-by-probe interaction were identified as candidates containing SFP. To address the quality of the bioinformatics approach, universal pyrosequencing assays were designed from Affymetrix exemplar sequences to independently assess polymorphisms within a subset of probes. Of those probes sampled from high-, medium-, and low-ranking categories, 20 of 27 were confirmed by pyrosequencing to contain SFPs. In most cases, the 25-mer probe sequence printed on the microarray diverged from Meishan, not occidental crosses. This analysis was used to define a set of highly reliable predicted SFPs according to their probability scores. Conclusions: By this method we detected transition and transversion single nucleotide polymorphisms, as well as insertions/deletions. These results demonstrate that this approach can identify polymorphisms between two breeds and/or lines of any species for which a short oligonucleotide array is available, and can be used to rapidly develop markers for genetic mapping and association analysis in species where high density genotyping platforms are otherwise unavailable. SNPs and INDELS discovered by this approach have been publicly deposited in NCBI’s SNP repository dbSNP. This method is an attractive bioinformatics tool for uncovering breed-by-probe interactions, for rapidly identifying expressed SNPs, for investigating potential functional correlations between gene expression and breed polymorphisms, and is robust enough to be used on any Affymetrix gene expression platform. Keywords: Transcriptional profiling of Day 25 porcine placentas

Dataset Information

Development of a versatile, target-oriented tiling microarray assay for measuring allele-specific gene expression

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure