Genome-wide analysis of allelic expression imbalance in human primary cells by high-throughput transcriptome resequencing.

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 approximately 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.

Fluorescence in situ hybridization with high-complexity repeat-free oligonucleotide probes generated by massively parallel synthesis.

The ability to visualize specific DNA sequences, on chromosomes and in nuclei, by fluorescence in situ hybridization (FISH) is fundamental to many aspects of genetics, genomics and cell biology. Probe selection is currently limited by the availability of DNA clones or the appropriate pool of DNA sequences for PCR amplification. Here, we show that liquid-phase probe pools from sequence capture technology can be adapted to generate fluorescently labelled pools of oligonucleotides that are very effective as repeat-free FISH probes in mammalian cells. As well as detection of small (15 kb) and larger (100 kb) specific loci in both cultured cells and tissue sections, we show that complex oligonucleotide pools can be used as probes to visualize features of nuclear organization. Using this approach, we dramatically reveal the disposition of exons around the outside of a chromosome territory core and away from the nuclear periphery.

A comprehensive resequence analysis of the KLK15-KLK3-KLK2 locus on chromosome 19q13.33.

Single nucleotide polymorphisms (SNPs) in the KLK3 gene on chromosome 19q13.33 are associated with serum prostate-specific antigen (PSA) levels. Recent genome wide association studies of prostate cancer have yielded conflicting results for association of the same SNPs with prostate cancer risk. Since the KLK3 gene encodes the PSA protein that forms the basis for a widely used screening test for prostate cancer, it is critical to fully characterize genetic variation in this region and assess its relationship with the risk of prostate cancer. We have conducted a next-generation sequence analysis in 78 individuals of European ancestry to characterize common (minor allele frequency, MAF >1%) genetic variation in a 56 kb region on chromosome 19q13.33 centered on the KLK3 gene (chr19:56,019,829-56,076,043 bps). We identified 555 polymorphic loci in the process including 116 novel SNPs and 182 novel insertion/deletion polymorphisms (indels). Based on tagging analysis, 144 loci are necessary to tag the region at an r (2) threshold of 0.8 and MAF of 1% or higher, while 86 loci are required to tag the region at an r (2) threshold of 0.8 and MAF >5%. Our sequence data augments coverage by 35 and 78% as compared to variants in dbSNP and HapMap, respectively. We observed six non-synonymous amino acid or frame shift changes in the KLK3 gene and three changes in each of the neighboring genes, KLK15 and KLK2. Our study has generated a detailed map of common genetic variation in the genomic region surrounding the KLK3 gene, which should be useful for fine-mapping the association signal as well as determining the contribution of this locus to prostate cancer risk and/or regulation of PSA expression.

In situ oligonucleotide synthesis on carbon materials: stable substrates for microarray fabrication.

Glass has become the standard substrate for the preparation of DNA arrays. Typically, glass is modified using silane chemistries to provide an appropriate functional group for nucleic acid synthesis or oligonucleotide immobilization. We have found substantial issues with the stability of these surfaces as manifested in the unwanted release of oligomers from the surface when incubated in aqueous buffers at moderate temperatures. To address this issue, we have explored the use of carbon-based substrates. Here, we demonstrate in situ synthesis of oligonucleotide probes on carbon-based substrates using light-directed photolithographic phosphoramidite chemistry and evaluate the stabilities of the resultant DNA arrays compared to those fabricated on silanized glass slides. DNA arrays on carbon-based substrates are substantially more stable than arrays prepared on glass. This superior stability enables the use of high-density DNA arrays for applications involving high temperatures, basic conditions, or where serial hybridization and dehybridization is desired.

Transcriptional changes in response to growth of Arabidopsis in high external calcium.

Transcriptional responses to growth in high environmental calcium concentrations were characterized and compared between wild-type and mutant Arabidopsis plants containing a knockout mutation in the gene encoding a cyclic nucleotide-gated channel (CNGC2). We show that the transcriptional profile of cngc2 plants grown in normal media resembled that from wild-type plants grown under elevated exogenous calcium conditions. The mutant grown in high-calcium media exhibited transcriptional changes not seen in the wild-type. The pattern of transcription suggests that adaptation to high external calcium overlaps with responses towards various biotic and abiotic stresses.

Identification of an in vitro transcription-based artifact affecting oligonucleotide microarrays.

This study identified the widely used T7 in vitro transcription system as a major source of artifact in the tiling array data from nine eukaryotic genomes. The most affected probes contained a sequence motif complementary to the +1 to +9 initial transcribed sequence (ITS) of the T7-(dT)(24) primer. The abundance of 5' ITS cRNA fragments produced during target preparation was sufficient to drive undesirable hybridization. A new T7-(dT)(24) primer with a modified ITS was designed that shifts the artifactual motifs as predicted and reduces the effect of the artifact. A computational algorithm was generated to filter out the likely artifactual probes from existing whole-genome tiling array data and improve probe selection. Further studies of Arabidopsis thaliana were conducted using both T7-(dT)(24) primers. While the artifact affected transcript discovery with tiling arrays, it showed only a minor impact on measurements of gene expression using commercially available 'gene-only' expression arrays.

Amplification and assembly of chip-eluted DNA (AACED): a method for high-throughput gene synthesis.

A basic problem in gene synthesis is the acquisition of many short oligonucleotide sequences needed for the assembly of genes. Photolithographic methods for the massively parallel synthesis of high-density oligonucleotide arrays provides a potential source, once appropriate methods have been devised for their elution in forms suitable for enzyme-catalyzed assembly. Here, we describe a method based on the photolithographic synthesis of long (>60mers) single-stranded oligonucleotides, using a modified maskless array synthesizer. Once the covalent bond between the DNA and the glass surface is cleaved, the full-length oligonucleotides are selected and amplified using PCR. After cleavage of flanking primer sites, a population of unique, internal 40mer dsDNA sequences are released and are ready for use in biological applications. Subsequent gene assembly experiments using this DNA pool were performed and were successful in creating longer DNA fragments. This is the first report demonstrating the use of eluted chip oligonucleotides in biological applications such as PCR and assembly PCR.

Higher-order looping and nuclear organization of Tcra facilitate targeted rag cleavage and regulated rearrangement in recombination centers.

V(D)J recombination is essential for generating a diverse array of B and T cell receptors that can recognize and combat foreign antigens. As with any recombination event, tight control is essential to prevent the occurrence of genetic anomalies that drive cellular transformation. One important aspect of regulation is directed targeting of the RAG recombinase. Indeed, RAG accumulates at the 3' end of individual antigen receptor loci poised for rearrangement; however, it is not known whether focal binding is involved in regulating cleavage, and what mechanisms lead to enrichment of RAG in this region. Here, we show that monoallelic looping out of the 3' end of the T cell receptor α (Tcra) locus, coupled with transcription and increased chromatin/nuclear accessibility, is linked to focal RAG binding and ATM-mediated regulation of monoallelic cleavage on looped-out 3' regions. Our data identify higher-order loop formation as a key determinant of directed RAG targeting and the maintenance of genome stability.

Whole exome capture in solution with 3 Gbp of data.

We have developed a solution-based method for targeted DNA capture-sequencing that is directed to the complete human exome. Using this approach allows the discovery of greater than 95% of all expected heterozygous singe base variants, requires as little as 3 Gbp of raw sequence data and constitutes an effective tool for identifying rare coding alleles in large scale genomic studies.

Genome-wide in situ exon capture for selective resequencing.

Increasingly powerful sequencing technologies are ushering in an era of personal genome sequences and raising the possibility of using such information to guide medical decisions. Genome resequencing also promises to accelerate the identification of disease-associated mutations. Roughly 98% of the human genome is composed of repeats and intergenic or non-protein-coding sequences. Thus, it is crucial to focus resequencing on high-value genomic regions. Protein-coding exons represent one such type of high-value target. We have developed a method of using flexible, high-density microarrays to capture any desired fraction of the human genome, in this case corresponding to more than 200,000 protein-coding exons. Depending on the precise protocol, up to 55-85% of the captured fragments are associated with targeted regions and up to 98% of intended exons can be recovered. This methodology provides an adaptable route toward rapid and efficient resequencing of any sizeable, non-repeat portion of the human genome.

Direct selection of human genomic loci by microarray hybridization.

We applied high-density microarrays to the enrichment of specific sequences from the human genome for high-throughput sequencing. After capture of 6,726 approximately 500-base 'exon' segments, and of 'locus-specific' regions ranging in size from 200 kb to 5 Mb, followed by sequencing on a 454 Life Sciences FLX sequencer, most sequence reads represented selection targets. These direct selection methods supersede multiplex PCR for the large-scale analysis of genomic regions.