Identification of novel fusion genes in lung cancer using breakpoint assembly of transcriptome sequencing data.
Bottom Line: Genomic translocation events frequently underlie cancer development through generation of gene fusions with oncogenic properties.Identification of such fusion transcripts by transcriptome sequencing might help to discover new potential therapeutic targets.We apply TRUP to RNA-seq data of different tumor types, and find it to be more sensitive than alternative tools in detecting chimeric transcripts, such as secondary rearrangements in EML4-ALK-positive lung tumors, or recurrent inactivating rearrangements affecting RASSF8.
Genomic translocation events frequently underlie cancer development through generation of gene fusions with oncogenic properties. Identification of such fusion transcripts by transcriptome sequencing might help to discover new potential therapeutic targets. We developed TRUP (Tumor-specimen suited RNA-seq Unified Pipeline) (https://github.com/ruping/TRUP), a computational approach that combines split-read and read-pair analysis with de novo assembly for the identification of chimeric transcripts in cancer specimens. We apply TRUP to RNA-seq data of different tumor types, and find it to be more sensitive than alternative tools in detecting chimeric transcripts, such as secondary rearrangements in EML4-ALK-positive lung tumors, or recurrent inactivating rearrangements affecting RASSF8.
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Mentions: Paired-end RNA-seq analysis of the EML4-ALK positive lung cancer cell-lines H3122 and H2228 revealed that in both cases EML4-ALK co-occurred with secondary in-frame chimeric transcripts: SOS1-ADCY3 in the case of H3122, and SND1-CFTR and DCBLD2-STXBP5L in the case of H2228 (Table 1; Figure 2a). We noticed that the genes involved in EML4-ALK and SOS1-ADCY3 were located in the same region of chromosome 2 (Figure 2b, upper panel). In fact, the arrangement of these two genes in the genome suggested that SOS1-ADCY3 might be generated by the same genomic event that had caused the EML4-ALK fusion. In order to test this hypothesis we first performed a break-apart FISH assay (ba-FISH) for both SOS1 and ADCY3 genes and a fusion assay for SOS1-ADCY3 on H3122 interphase chromosomes, to test whether the alteration happened at the genomic level (Additional file 4). We then performed ba-FISH for both ALK and ADCY3 separately, on metaphase chromosomes of the same cell line (Figure 2b, lower panel): in the case of ADCY3 ba-FISH we found one aberrant single green signal with loss of the correspondent red signal. The same pattern was observed when performing the assay for ALK. We therefore reasoned that if both rearrangements were linked, when performing both assays together we should see the same pattern as observed separately (that is, one single green signal), since the two green signals would overlap and therefore be indistinguishable (Additional file 5, arrow A). On the contrary, if the two rearrangements occurred on different alleles, we should be able to distinguish two separate single green signals, one from the assay testing ALK and one for the assay assessing ADCY3 (Additional file 5, arrow B). The combined ALK-ADCY3 assay only generated one single green signal suggesting that the two rearrangements were likely to be physically linked (Figure 2b, lower panel). In addition to these two cell-lines, we validated at least one secondary in-frame chimeric transcript in four additional EML4-ALK positive primary tumors: SNAP29-CELSR1 and PIGF-CHMP3 in sample S00054; MYO10-GPC5 and ARHGEF7-ZDHHC11 in S01122; TAF4-LSM14B in S01124; and NUP85-GPC3 in S01320 (Table 1).Figure 2