Targeted next-generation sequencing of a cancer transcriptome enhances detection of sequence variants and novel fusion transcripts

Abstract

Targeted RNA-Seq combines next-generation sequencing with capture of sequences from a relevant subset of a transcriptome. When testing by capturing sequences from a tumor cDNA library by hybridization to oligonucleotide probes specific for 467 cancer-related genes, this method showed high selectivity, improved mutation detection enabling discovery of novel chimeric transcripts, and provided RNA expression data. Thus, targeted RNA-Seq produces an enhanced view of the molecular state of a set of "high interest" genes.

Figure 1

Increase in sequence coverage. (a) Mean sequence coverage by region. Transcript regions (5' UTR, CDS, 3' UTR) were divided into deciles, and the sequence coverage for each decile was averaged across all 887 target transcripts. Coverage is displayed for before hybrid selection (blue) and after hybrid selection (red). The average length of each region is 292, 2,136, and 1,729, respectively. (b) Distribution of sequence coverage for targeted genes. For each sequence-coverage threshold (x-axis), the fraction of 467 genes at or above that threshold is plotted (y-axis) for before hybrid selection (blue) and after hybrid selection (red).

Figure 2

Preservation of expression levels of target transcripts in hybrid selection. For each target transcript, the sequence coverage of the coding region is plotted before and after hybrid selection.

Figure 3

Sequences from NUP214-XKR3 fusion transcripts detected after hybrid selection. After hybrid selection, 152 reads were aligned to the transcriptome and detected as NUP214-XKR3 fusions. From top to bottom, we observed 137, four, eight, and three reads for these transcripts. The NUP214 (exon 27) to XKR3 (exon 4) has a stop codon downstream (not shown). Only NUP214 (exon 29) to XKR3 (exon 4) retains an open reading frame downstream of the fusion. Before hybrid selection, eight reads were aligned to the transcriptome and detected as NUP214-XKR3 fusions; only the NUP214 (exon 29) to XKR3 (exon 2) transcript was detected. Sequence from NUP214 DNA is shown as lower case, and from XKR3, as bold and upper case.