Exome RNA sequencing reveals rare and novel alternative transcripts

Abstract

RNA sequencing has become an important method to perform hypothesis-free characterization of global gene expression. One of the limitations of RNA sequencing is that most sequence reads represent highly expressed transcripts, whereas low level transcripts are challenging to detect. To combine the benefits of traditional expression arrays with the advantages of RNA sequencing, we have used whole exome enrichment prior to sequencing of total RNA. We show that whole exome capture can be successfully applied to cDNA to study the transcriptional landscape in human tissues. By introducing the exome enrichment step, we are able to identify transcripts present at very low levels, which are below the level of detection in conventional RNA sequencing. Although the enrichment increases the ability to detect presence of transcripts, it also lowers the accuracy of quantification of expression levels. Our results yield a large number of novel exons and splice isoforms, suggesting that conventional RNA sequencing methods only detect a small fraction of the full transcript diversity. We propose that whole exome enrichment of RNA is a suitable strategy for genome-wide discovery of novel transcripts, alternative splice variants and fusion genes.

RNA sequencing unravels the genetics of refractory/relapsed T-cell acute lymphoblastic leukemia. Prognostic and therapeutic implications

“…We identified 183 predicted fusion transcripts (median 6/sample; range, 0-36) (Online Supplementary Table S6A) predominantly involving genes localized next to each other on the same chromosome, and likely representing read-through of transcription. 15,20 For example, two fusion transcripts involving adjacent genes, namely SMG5-PAQR6 (n=2) and TTTY15-USP9Y (n=2), previously described in prostatic cancer [21][22][23] and also identified in two cases of our validation cohort, were detected in normal thymus cells from healthy donors.…”

“…Along this line, significantly shorter overall survival, disease-free survival and event-free survival were observed in patients harboring JAK/STAT mutations compared to patients without alterations in the pathways (Figure 4A-C). In detail, the overall, disease-free and eventfree survival probabilities were 0% at 20 …”

Molecular indexing enables quantitative targeted RNA sequencing and reveals poor efficiencies in standard library preparations

“…Reports estimate that ∼40 million reads may be required for the reliable measurement of gene expression for transcripts of high and moderate abundance, and as many as 500 million reads may be required to cover the full sequence diversity of a complex transcript library (1,5,6). In routine use, sparse coverage is further compromised by the practice of multiplexing samples in a single RNA-Seq run, primarily driven by cost constraints when designing studies involving large numbers of samples, such as those required in clinical applications.To efficiently sample the rare or low-abundance isoforms of transcripts, capture methods have recently been used (7,8). These promising methods use a targeted strategy whereby genomic regions of interest are enriched through hybridization capture and amplification before sequence sampling.…”

“…To efficiently sample the rare or low-abundance isoforms of transcripts, capture methods have recently been used (7,8). These promising methods use a targeted strategy whereby genomic regions of interest are enriched through hybridization capture and amplification before sequence sampling.…”

“…When applied to generate the sequencing library for RNA-Seq, the method can effectively enrich rare transcripts and reduce inefficiencies associated with sampling large numbers of abundant transcripts present in an unmodified library. However, because targeted sequencing procedures introduce biases caused by unequal capture and amplification efficiencies across different genes, accurate gene expression measurements cannot be assured (7).…”