Human transcriptome array for high-throughput clinical studies

Abstract

A 6.9 million-feature oligonucleotide array of the human transcriptome [Glue Grant human transcriptome (GG-H array)] has been developed for high-throughput and cost-effective analyses in clinical studies. This array allows comprehensive examination of gene expression and genome-wide identification of alternative splicing as well as detection of coding SNPs and noncoding transcripts. The performance of the array was examined and compared with mRNA sequencing (RNA-Seq) results over multiple independent replicates of liver and muscle samples. Compared with RNA-Seq of 46 million uniquely mappable reads per replicate, the GG-H array is highly reproducible in estimating gene and exon abundance. Although both platforms detect similar expression changes at the gene level, the GG-H array is more sensitive at the exon level. Deeper sequencing is required to adequately cover low-abundance transcripts. The array has been implemented in a multicenter clinical program and has generated high-quality, reproducible data. Considering the clinical trial requirements of cost, sample availability, and throughput, the GG-H array has a wide range of applications. An emerging approach for large-scale clinical genomic studies is to first use RNA-Seq to the sufficient depth for the discovery of transcriptome elements relevant to the disease process followed by high-throughput and reliable screening of these elements on thousands of patient samples using custom-designed arrays.

Fig. 1.

Design of the GG-H human transcriptome array for comprehensive examination of gene and exon expression, alternative splicing, and additional contents of human transcriptome. (A) On a 3′ gene array, such as the Affymetrix HU-133 Array, 11 probes were designed for the 3′-end exon(s) of each gene. On an exon array, such as the Affymetrix Human Exon 1.0 ST Array, there are two to four probes for each exon of the gene. In contrast, the GG-H array uses, on average, 10 probes for each exon/PSR and 4 probes for each exon–exon junction; in addition, 6 pairs of probes were designed for each coding SNP, and 10 probes were designed for each noncoding RNA transcript. (B) Comparison of the GG-H array contents with mRNA sequencing data on multiple tissues. The percentages of exons, junctions, UTUs, and ncRNAs (y axis) supported by at least a specified number of sequencing reads (x axis) are shown.

Fig. 2.

Comparison of the performances of the GG-H array and mRNA sequencing. (A) Reproducibility of exon and gene expression measured by array and sequencing. In each panel, scatter plots of logged expression values between four independent replicates of a reference muscle sample are shown in the bottom left corner and in the top right corner, the corresponding Pearson correlation coefficients are shown. (B) Comparison of differentially expressed exons and genes identified by GG-H array and mRNA sequencing.

Fig. 3.

Detection of alternative splicing events using exon and junction probes on the array. Two isoforms of SLK are alternatively spliced between liver and muscle; the green lines represent an isoform-skipping exon 15 (ENST00000335753), and the blue lines represent another isoform including exon 15 (ENST00000369755). (A) Changes of the signal of junction and exon probes. (B) Changes of the calculated expression of exons and junctions. Exon expression is shown at the diagonal, and junction expression is shown off the diagonal of two connecting exons.