Fusion transcripts are hybrid RNAs composed of transcripts from two separate genes. Such chimeric RNAs often result from gene fusions and are associated with cancer. Several classic fusions like BCR-ABL have been successfully targeted for cancer therapy. While fusions are often caused by DNA rearrangements, they can also be produced by RNA splicing of neighboring genes. Next generation sequencing, especially RNA-Seq, allows detection of fusion transcripts and has led to the development of many computational tools to identify fusions from sequencing data.

1. Nalesh Jadhav
RNA Fusion Transcripts

2. ● Fusion Transcript: Hybrid RNA, composed of transcripts of two separate genes
● Such chimeric RNAs were known to be the result of gene fusions and are associated with cancer. Example, gene fusions like
BCR-ABL are found in many tumors.
● These fusions are good targets for cancer diagnosis and therapeutic applications. Several classic fusions, such as: BCR-ABL,
PML-RAR, and EML4-ALK, are the poster children for successful targeted cancer therapy
● Even though chimeric RNAs are mostly known to be the products of chromosomal rearrangement at the DNA level, fusion
transcripts can also be produced by trans-splicing and cis-splicing between neighboring genes.
What is a Fusion Transcript?
Source: doi: 10.1002/wrna.1382 PMID: 27485475 Identifying Fusion Transcripts Using Next Generation Sequencing Advanced Review

3. What is a Fusion Transcript?
● With the advancement of Next Generation Sequencing (NGS), fusions can be detected either in genomic DNA sequencing
datasets or transcriptome sequencing (RNA-Seq) datasets
● In 2010, first dedicated tool for the fusion detection (i.e. FusionSeq) was published. Since 2010, around 33 computational
tools have been developed for detecting fusion transcripts using RNA-Seq data
Source: doi: 10.1002/wrna.1382 PMID: 27485475 Identifying Fusion Transcripts Using Next Generation Sequencing Advanced Review

4. Role of Fusion Transcript in Cancer?
Source: First published: 13 August 2019 https://doi.org/10.1002/wrna.1562

5. ONCOGENIC FUSION TRANSCRIPTS—PARTNERS IN CANCER
● BCR-ABL1 in leukemia
● RUNX1-RUNX1T1 in acute myeloid leukemia
● EML4-ALK in non-small cell lung cancer
● NUT-fusions in NUT-midline carcinoma
● TMPRSS2-ERG in prostate cancer
● EWS-FLI1 and EWS-ERG in Ewing sarcoma
Received: 6 May 2019 Revised: 5 July 2019 Accepted: 8 July 2019 DOI: 10.1002/wrna.1562 Fusion transcripts: Unexploited vulnerabilities in
cancer?

6. WGS or RNA-Seq?
● WGS: requires a great amount of sequencing, and
exhaustive computational analysis
● The cost of WGS of human samples is generally higher
● WGS will only detect fusion events that occur at the DNA
level. This is a limitation, as WGS will miss all of the
fusion events that occur at the RNA splicing level.
● However, this feature could be desirable in cases where
researchers only want to find subset of fusions that are
generated by chromosomal rearrangement.
● RNA-Seq sequencing only sequences a small part (~2%)
of the genome that is transcribed and spliced into mature
mRNA.
● RNA-Seq will detect ‘intergenically’ spliced fusions that
only occur at the RNA level.
● RNA-Seq also allows for the detection of multiple
alternative splice variants resulting from fusions.
● Low cost and quick turnaround time make RNA-Seq very
popular in fusion transcript studies.

7. Detection of Fusion Transcript:
The flow of fusion detection can be divided into three steps:
● Reads mapping and filtering,
● Fusion junction detection, and
● Fusion assembly and selection
(i) Paired-end reads are mapped to reference sequences
and discordantly mapped reads (spanning/improperly
reads) are directly aligned to the target fusion genes.
(ii) For paired-end reads with one or both ends unaligned
(potential split reads), the unmapped mate is cut into several
pieces to be aligned to estimated fusion boundaries. Here,
the two pieces of same read are connected by dashed line.
(iii) Fusion candidate sequences are assembled.
(iv) Assembled fusion sequences with highest probability to
be selected as real fusion. Vertical dotted line represents the
fusion junction.
Source: doi: 10.1002/wrna.1382 PMID: 27485475 Identifying Fusion Transcripts Using Next Generation Sequencing Advanced Review

8. Tools for RNA Fusion Transcript detection:

9. JAFFA1.09 - High sensitivity transcriptome-focused fusion gene detection (reads of 100 bp or greater), a multi-
step pipeline that takes either raw RNA-Seq reads, or pre-assembled transcripts, then searches for gene fusions. It
will output the names and locations of candidate gene fusions along with the cDNA sequence of their breakpoints
FusionCatcher 1.20 - a tool for finding somatic fusion genes in paired-end RNA-sequencing data
Recommendations to detect fusion events are to: 1) use paired-end format 2) perform RNA-Seq if interested in
fusion transcripts not limited to chromosomal rearrangement 3) achieve reasonable reads length (>70bp) 4)
achieve reasonable reads depth (>50 million) and 5) select appropriate software based on needs.
Arriba 1.1.0 - It is based on the ultrafast STAR aligner and the post-alignment runtime is very less. Apart from
gene fusions, Arriba can detect other structural rearrangements with potential clinical relevance, such as exon
duplications or truncations of genes (i.e., breakpoints in introns and intergenic regions)
Tools to be explored: