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Digital RNA allelotyping reveals tissue-specific and allele-specific gene expression in human.

Zhang K, Li JB, Gao Y, Egli D, Xie B, Deng J, Li Z, Lee JH, Aach J, Leproust EM, Eggan K, Church GM - Nat. Methods (2009)

Bottom Line: Approximately 11-22% of the heterozygous mRNA-associated SNPs showed allele-specific expression in each cell line and 4.3-8.5% were tissue-specific, suggesting the presence of tissue-specific cis regulation.We found that the variation of allelic ratios in gene expression among different cell lines was primarily explained by genetic variations, much more so than by specific tissue types or growth conditions.Comparison of expressed SNPs on the sense and antisense transcripts suggested that allelic ratios are primarily determined by cis-regulatory mechanisms on the sense transcripts.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, University of California at San Diego, La Jolla, California, USA. kzhang@bioeng.ucsd.edu

ABSTRACT
We developed a digital RNA allelotyping method for quantitatively interrogating allele-specific gene expression. This method involves ultra-deep sequencing of padlock-captured single-nucleotide polymorphisms (SNPs) from the transcriptome. We characterized four cell lines established from two human subjects in the Personal Genome Project. Approximately 11-22% of the heterozygous mRNA-associated SNPs showed allele-specific expression in each cell line and 4.3-8.5% were tissue-specific, suggesting the presence of tissue-specific cis regulation. When we applied allelotyping to two pairs of sibling human embryonic stem cell lines, the sibling lines were more similar in allele-specific expression than were the genetically unrelated lines. We found that the variation of allelic ratios in gene expression among different cell lines was primarily explained by genetic variations, much more so than by specific tissue types or growth conditions. Comparison of expressed SNPs on the sense and antisense transcripts suggested that allelic ratios are primarily determined by cis-regulatory mechanisms on the sense transcripts.

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Digital allelotyping with padlock probes. (a) The design of padlock probe (top) and a schematic diagram of padlock capturing experiments (bottom). (b) (c) The experimental and analytic workflow of digital allelotyping assay.
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Figure 1: Digital allelotyping with padlock probes. (a) The design of padlock probe (top) and a schematic diagram of padlock capturing experiments (bottom). (b) (c) The experimental and analytic workflow of digital allelotyping assay.

Mentions: We designed single-stranded DNA probes to capture SNPs from the human genome and transcriptome for sequencing (Fig. 1a). Each probe contains two terminal capturing arms (H1 and H2), that can anneal to the flanking region of the targeted SNPs with a gap of one or more nucleotide bases. In the capturing reaction similar to Molecular Inversion Probes12, the gap is filled by a DNA polymerase and closed by a DNA thermal ligase. The capturing arms are connected by a common linker DNA sequence. This linker contains priming sites for multiplex PCR amplification of the circularized single-stranded DNA probes. After the circularization reaction and PCR amplification, the resulting libraries are sequenced with Illumina Genome Analyzer. As demonstrated previously, circularization of padlock probes is extremely specific; >10,000 targets can be captured simultaneously in a single tube12–15.


Digital RNA allelotyping reveals tissue-specific and allele-specific gene expression in human.

Zhang K, Li JB, Gao Y, Egli D, Xie B, Deng J, Li Z, Lee JH, Aach J, Leproust EM, Eggan K, Church GM - Nat. Methods (2009)

Digital allelotyping with padlock probes. (a) The design of padlock probe (top) and a schematic diagram of padlock capturing experiments (bottom). (b) (c) The experimental and analytic workflow of digital allelotyping assay.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2742772&req=5

Figure 1: Digital allelotyping with padlock probes. (a) The design of padlock probe (top) and a schematic diagram of padlock capturing experiments (bottom). (b) (c) The experimental and analytic workflow of digital allelotyping assay.
Mentions: We designed single-stranded DNA probes to capture SNPs from the human genome and transcriptome for sequencing (Fig. 1a). Each probe contains two terminal capturing arms (H1 and H2), that can anneal to the flanking region of the targeted SNPs with a gap of one or more nucleotide bases. In the capturing reaction similar to Molecular Inversion Probes12, the gap is filled by a DNA polymerase and closed by a DNA thermal ligase. The capturing arms are connected by a common linker DNA sequence. This linker contains priming sites for multiplex PCR amplification of the circularized single-stranded DNA probes. After the circularization reaction and PCR amplification, the resulting libraries are sequenced with Illumina Genome Analyzer. As demonstrated previously, circularization of padlock probes is extremely specific; >10,000 targets can be captured simultaneously in a single tube12–15.

Bottom Line: Approximately 11-22% of the heterozygous mRNA-associated SNPs showed allele-specific expression in each cell line and 4.3-8.5% were tissue-specific, suggesting the presence of tissue-specific cis regulation.We found that the variation of allelic ratios in gene expression among different cell lines was primarily explained by genetic variations, much more so than by specific tissue types or growth conditions.Comparison of expressed SNPs on the sense and antisense transcripts suggested that allelic ratios are primarily determined by cis-regulatory mechanisms on the sense transcripts.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, University of California at San Diego, La Jolla, California, USA. kzhang@bioeng.ucsd.edu

ABSTRACT
We developed a digital RNA allelotyping method for quantitatively interrogating allele-specific gene expression. This method involves ultra-deep sequencing of padlock-captured single-nucleotide polymorphisms (SNPs) from the transcriptome. We characterized four cell lines established from two human subjects in the Personal Genome Project. Approximately 11-22% of the heterozygous mRNA-associated SNPs showed allele-specific expression in each cell line and 4.3-8.5% were tissue-specific, suggesting the presence of tissue-specific cis regulation. When we applied allelotyping to two pairs of sibling human embryonic stem cell lines, the sibling lines were more similar in allele-specific expression than were the genetically unrelated lines. We found that the variation of allelic ratios in gene expression among different cell lines was primarily explained by genetic variations, much more so than by specific tissue types or growth conditions. Comparison of expressed SNPs on the sense and antisense transcripts suggested that allelic ratios are primarily determined by cis-regulatory mechanisms on the sense transcripts.

Show MeSH