LAUSR

The KIAA1549:BRAF fusion is the most common alteration in pilocytic astrocytoma (PA). It is generated by a focal tandem duplication at 7q34 and acts as an oncogene by driving the mitogen-activated protein kinase (MAPK) pathway [4]. Detection of this characteristic genetic event is of high clinical relevance, both for its diagnostic/prognostic relevance and as a therapeutic target. RNA sequencing (RNASeq) of fresh-frozen or formalin-fixed paraffin-embedded (FFPE) tissue has recently gained popularity in the diagnostic setting [1]. By identifying split reads that map to two different genomic loci, RNA-Seq data can be used to detect expressed fusion genes. Several tools have been developed for this purpose, including Arriba (https ://githu b.com/suhri g/arrib a), FusionCatcher (https ://githu b.com/ndani el/fusio ncatc her) and STAR-Fusion (https ://githu b.com/STAR-Fusio n/STAR-Fusio n). Previous studies have suggested that the KIAA1549:BRAF fusion is expressed at a low level [5–7], but the reliability of detection of this important fusion using different RNA-Seq analysis pipelines has not been examined so far. To this end, we generated RNA-Seq data (polyAenriched, TruSeq Stranded, 2 × 100 bp paired-end reads) from 22 fresh-frozen pediatric PA tumor samples, in which a KIAA1549:BRAF fusion had previously been identified by whole-genome sequencing (WGS) [3]. The raw data was subsequently aligned by STAR [2] (v2.7.3a), and gene fusions were identified using Arriba (v1.1.0). Despite a total read count of about 200 million reads per sample (Fig. 1a), the KIAA1549:BRAF fusion was only correctly identified in 14/22 samples (Fig. 1b). In three additional samples, Arriba had identified but then discarded the fusion, as it was supported by just one sequencing read. In five samples, the fusion was not detected at all with this workflow. To investigate the influence of sequencing depth, we re-sequenced these five samples, substantially Electronic supplementary material The online version of this article (https ://doi.org/10.1007/s0040 1-020-02167 -1) contains supplementary material, which is available to authorized users.