ASEtrap: A biological method for speeding up the exploration of spliceomes

Abstract

Alternative splicing (AS) of pre-messenger RNA is a major mechanism for generating protein diversity from a limited number of genes in higher eukaryotes, and it constitutes a central mode of genetic regulation. Thus, efficient methods are needed to systematically identify new AS events at a genomic scale across different tissues, stages of development, and physiological or pathological conditions in order to better understand gene expression. To fulfill this goal, we have designed the ASEtrap, which is a cloning procedure for producing AS libraries that is based on a single-stranded trap consisting of an ssDNA-binding protein. In this paper, we have applied our approach to the construction of an AS library and a Control library from human placenta. By analyzing 9226 and 9999 sequences of the AS and Control libraries, respectively, we show that internal AS events (events that can be identified by the sole resources provided by either the AS or the Control library) and the discovery rate of new AS events measured at early stages of sequencing were nine to 10 times higher in the former than in the latter. Moreover, by performing a search for new AS events within a group of 162 known drug target genes, we identified six new events in six genes, and we observed that they all were discovered exclusively through the AS library. Thus, it appears that ASEtrap has the potential to greatly facilitate the determination of the total complement of splice variants expressed in human, as well as other organisms.

Annotating genomes with massive-scale RNA sequencing

“…However, as we build all possible models that correspond to the longest possible paths going from one covtig to another through validated junctions, some of the models probably do not correspond to real transcripts (for instance, if they link alternative exons that are incompatible, like models M 3 and M 4 in Figure 1 ). Since the long-range splice contiguity can not be inferred from short reads, we quantified short-range alternative splicing events in the models (all models, and only CDS portions of coding models) and in the cDNAs [ 46 ] (Table 4 ).…”

“…Additionally, the redundancy was removed from the cDNAs by discarding all transcript structures that were fully included in longer structures. We detected all pairwise alternative splicing events between intron pairs, with the same method as described in [ 46 ]. All tandemly duplicated genes were discarded from the alternative splicing events detected, since such genes may be artificially linked by cDNA mapping as well as model construction, and would generate false alternative splice forms spanning several loci instead of one.…”

The genome sequence of the model ascomycete fungus Podospora anserina

“…These transcript models were fused by a single linkage clustering approach, in which transcripts from the same genomic region sharing at least 100 bp are merged [ 98 ]. These clusters were used to detect alternative splicing events [ 99 ].…”

Alternative splicing and breast cancer

“…Therefore, other high-throughput technologies allowing to identify new splicing variants are required. 67,68 In this context, deep sequencing or RNA-seq (Fig. 4) should speed up the identification of new splicing variants.…”