Human Molecular Genetics Advance Access originally published online on October 13, 2009
Human Molecular Genetics 2010 19(1):122-134; doi:10.1093/hmg/ddp473
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Genome-wide analysis of allelic expression imbalance in human primary cells by high-throughput transcriptome resequencing
1 Centre for Digestive Diseases, Blizard Institute of Cell and Molecular Science, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London E1 2AT, UK, 2 Department of Medical Genetics, Juvenile Diabetes Research Foundation/Wellcome Trust Diabetes and Inflammation Laboratory, Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 0XY, UK, 3 Genome Centre, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, UK and 4 Roche NimbleGen, 504 S. Rosa Rd. Madison, WI 35393
* To whom correspondence should be addressed. Tel: +44 1223762107; Fax: +44 1223762102; Email: vincent.plagnol{at}cimr.cam.ac.uk
Received May 14, 2009; Revised September 17, 2009; Accepted October 9, 2009
Many disease-associated variants identified by genome-wide association (GWA) studies are expected to regulate gene expression. Allele-specific expression (ASE) quantifies transcription from both haplotypes using individuals heterozygous at tested SNPs. We performed deep human transcriptome-wide resequencing (RNA-seq) for ASE analysis and expression quantitative trait locus discovery. We resequenced double poly(A)-selected RNA from primary CD4+ T cells (n = 4 individuals, both activated and untreated conditions) and developed tools for paired-end RNA-seq alignment and ASE analysis. We generated an average of 20 million uniquely mapping 45 base reads per sample. We obtained sufficient read depth to test 1371 unique transcripts for ASE. Multiple biases inflate the false discovery rate which we estimate to be 
50% for random SNPs. However, after controlling for these biases and considering the subset of SNPs that pass HapMap QC, 4.6% of heterozygous SNP-sample pairs show evidence of imbalance (P < 0.001). We validated four findings by both bacterial cloning and Sanger sequencing assays. We also found convincing evidence for allelic imbalance at multiple reporter exonic SNPs in CD6 for two samples heterozygous at the multiple sclerosis-associated variant rs17824933, linking GWA findings with variation in gene expression. Finally, we show in CD4+ T cells from a further individual that high-throughput sequencing of genomic DNA and RNA-seq following enrichment for targeted gene sequences by sequence capture methods offers an unbiased means to increase the read depth for transcripts of interest, and therefore a method to investigate the regulatory role of many disease-associated genetic variants.
These authors contributed equally to this work.