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Allele-specific expression assays using Solexa.

Main BJ, Bickel RD, McIntyre LM, Graze RM, Calabrese PP, Nuzhdin SV - BMC Genomics (2009)

Bottom Line: To validate this method, we measured the allelic bias in a dilution series and found high correlations between measured and expected values (r>0.9, p < 0.001).We applied this method to a set of 5 genes in a Drosophila simulans parental mix, F1 and introgression and found that for these genes the majority of expression divergence can be explained by cis-regulatory variation.This will be a valuable technique for molecular and population genetic studies, as well as for verification of genome-wide data sets.

View Article: PubMed Central - HTML - PubMed

Affiliation: Section of Molecular and Computational Biology, Department of Biological Sciences, University of Southern California, Los Angeles, California 90089, USA. bmain@usc.edu

ABSTRACT

Background: Allele-specific expression (ASE) assays can be used to identify cis, trans, and cis-by-trans regulatory variation. Understanding the source of expression variation has important implications for disease susceptibility, phenotypic diversity, and adaptation. While ASE is commonly measured via relative fluorescence at a SNP, next generation sequencing provides an opportunity to measure ASE in an accurate and high-throughput manner using read counts.

Results: We introduce a Solexa-based method to perform large numbers of ASE assays using only a single lane of a Solexa flowcell. In brief, transcripts of interest, which contain a known SNP, are PCR enriched and barcoded to enable multiplexing. Then high-throughput sequencing is used to estimate allele-specific expression using sequencing counts. To validate this method, we measured the allelic bias in a dilution series and found high correlations between measured and expected values (r>0.9, p < 0.001). We applied this method to a set of 5 genes in a Drosophila simulans parental mix, F1 and introgression and found that for these genes the majority of expression divergence can be explained by cis-regulatory variation.

Conclusion: We present a new method with the capacity to measure ASE for large numbers of assays using as little as one lane of a Solexa flowcell. This will be a valuable technique for molecular and population genetic studies, as well as for verification of genome-wide data sets.

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Verification of ASE assays using Solexa. Each data point represents one of three replicate dilutions analyzed at each step of the dilution series (9:1, 8:2, 7:3, 5:5, 3:7, 2:8, 1:9). The distribution of sequencing reads within each gene is demonstrated as follows: mean ± SE (n = 21). DSX = 11.3 ± 3.8, CG2604 = 2,478.3 ± 288.8, CG10824 = 2,756.4 ± 333.5, CG11459 = 11,578.6 ± 2515.1.
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Figure 4: Verification of ASE assays using Solexa. Each data point represents one of three replicate dilutions analyzed at each step of the dilution series (9:1, 8:2, 7:3, 5:5, 3:7, 2:8, 1:9). The distribution of sequencing reads within each gene is demonstrated as follows: mean ± SE (n = 21). DSX = 11.3 ± 3.8, CG2604 = 2,478.3 ± 288.8, CG10824 = 2,756.4 ± 333.5, CG11459 = 11,578.6 ± 2515.1.

Mentions: To verify the accuracy of Solexa and the normalization procedure, we created three replicate dilution series using genomic DNA from the tester line and an experimental line (W line 84). Then we estimated the allelic bias at each step of the dilution (in multiplex) using a separate sequencing lane. All four genes demonstrated a strong correlation (r >0.9 p < 0.001) between the expected and observed allelic bias (Figure 4). The gene dsx had a relatively low correlation, because there was very limited sequencing coverage for all samples within this gene. Thus, the Solexa sequencing output can be used to accurately measure the relative abundance of alleles in a given sample.


Allele-specific expression assays using Solexa.

Main BJ, Bickel RD, McIntyre LM, Graze RM, Calabrese PP, Nuzhdin SV - BMC Genomics (2009)

Verification of ASE assays using Solexa. Each data point represents one of three replicate dilutions analyzed at each step of the dilution series (9:1, 8:2, 7:3, 5:5, 3:7, 2:8, 1:9). The distribution of sequencing reads within each gene is demonstrated as follows: mean ± SE (n = 21). DSX = 11.3 ± 3.8, CG2604 = 2,478.3 ± 288.8, CG10824 = 2,756.4 ± 333.5, CG11459 = 11,578.6 ± 2515.1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2749874&req=5

Figure 4: Verification of ASE assays using Solexa. Each data point represents one of three replicate dilutions analyzed at each step of the dilution series (9:1, 8:2, 7:3, 5:5, 3:7, 2:8, 1:9). The distribution of sequencing reads within each gene is demonstrated as follows: mean ± SE (n = 21). DSX = 11.3 ± 3.8, CG2604 = 2,478.3 ± 288.8, CG10824 = 2,756.4 ± 333.5, CG11459 = 11,578.6 ± 2515.1.
Mentions: To verify the accuracy of Solexa and the normalization procedure, we created three replicate dilution series using genomic DNA from the tester line and an experimental line (W line 84). Then we estimated the allelic bias at each step of the dilution (in multiplex) using a separate sequencing lane. All four genes demonstrated a strong correlation (r >0.9 p < 0.001) between the expected and observed allelic bias (Figure 4). The gene dsx had a relatively low correlation, because there was very limited sequencing coverage for all samples within this gene. Thus, the Solexa sequencing output can be used to accurately measure the relative abundance of alleles in a given sample.

Bottom Line: To validate this method, we measured the allelic bias in a dilution series and found high correlations between measured and expected values (r>0.9, p < 0.001).We applied this method to a set of 5 genes in a Drosophila simulans parental mix, F1 and introgression and found that for these genes the majority of expression divergence can be explained by cis-regulatory variation.This will be a valuable technique for molecular and population genetic studies, as well as for verification of genome-wide data sets.

View Article: PubMed Central - HTML - PubMed

Affiliation: Section of Molecular and Computational Biology, Department of Biological Sciences, University of Southern California, Los Angeles, California 90089, USA. bmain@usc.edu

ABSTRACT

Background: Allele-specific expression (ASE) assays can be used to identify cis, trans, and cis-by-trans regulatory variation. Understanding the source of expression variation has important implications for disease susceptibility, phenotypic diversity, and adaptation. While ASE is commonly measured via relative fluorescence at a SNP, next generation sequencing provides an opportunity to measure ASE in an accurate and high-throughput manner using read counts.

Results: We introduce a Solexa-based method to perform large numbers of ASE assays using only a single lane of a Solexa flowcell. In brief, transcripts of interest, which contain a known SNP, are PCR enriched and barcoded to enable multiplexing. Then high-throughput sequencing is used to estimate allele-specific expression using sequencing counts. To validate this method, we measured the allelic bias in a dilution series and found high correlations between measured and expected values (r>0.9, p < 0.001). We applied this method to a set of 5 genes in a Drosophila simulans parental mix, F1 and introgression and found that for these genes the majority of expression divergence can be explained by cis-regulatory variation.

Conclusion: We present a new method with the capacity to measure ASE for large numbers of assays using as little as one lane of a Solexa flowcell. This will be a valuable technique for molecular and population genetic studies, as well as for verification of genome-wide data sets.

Show MeSH