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  • Because most fusion detection methods

    2020-10-16

    Because most fusion detection methods were developed to analyze paired-end reads (and especially Illumina data), they are not suitable for identifying fusion junction-spanning reads in single-end Ion Torrent libraries. Therefore, we developed the FusionDetect algorithm (implemented in the ChildDecode software) for the detection of fusion junction-spanning reads in our Ion Torrent data. By using a high-speed, scalable, cloud-based bioinformatics platform, we believe we can achieve rapid turnaround without any upfront investment in computing infrastructure. From our experience, analysis took <1 minute per sample (using the Amazon Elastic Compute Cloud web service). The coupling of a cloud-based bioinformatics tool (ChildDecode) with ChildSeq-RNA should alleviate computational Dyphylline australia associated with NGS-based assays. Therefore, although the NGS components of ChildSeq-RNA are likely limited to centers with an appropriate technology base, the use of a cloud-based system for downstream data analysis should allow for greater extension of this platform to centers with less computing capacity. Moreover, because most of the technologies used in this study are on the basis of well-established NGS platforms, the incorporation of ChildSeq-RNA into any small- to medium-sized facility undertaking NGS-based diagnostics should become increasingly feasible. Recently, Sweeney et al published a study using whole-transcriptome sequencing (with Illumina MiSeq and HiSeq machines) to detect gene fusions in sarcomas (including ES). They were able to successfully identify fusions using FFPE material. Both our studies demonstrate that limited fusion-supporting reads (from 1 to 35 reads in their study, and from 1 to 29 reads in ours) can be used to identify a fusion transcript. However, Sweeney et al showed that, when using a whole-transcriptome sequencing approach, sufficient sequencing depth is required to detect fusion transcripts. For example, Sweeney et al found that their method was unable to detect fusions in ES samples using an MiSeq (a smaller, benchtop Illumina sequencer with much lower capacity than an HiSeq), but could do so using an HiSeq. Thus, their method relies on high-capacity sequencers (eg, HiSeq), which small- and medium-sized laboratories may not have access to. The main difference between our ChildSeq-RNA protocol and the protocol used by Sweeney et al is the use of a molecular target-capture step in our protocol that enriches RNA material from known translocation-associated genes. This additional step enables fusion transcript detection with less read output (using smaller sequencers, such as an MiSeq or IonT PGM, as in our study), thus potentially rendering our method more feasible for small- and medium-sized laboratories. More important, both our study and the study by Sweeney et al demonstrate the potential of NGS-based assays for molecular diagnostics.
    Acknowledgments
    Introduction Ewing sarcoma/primitive neuroectodermal tumor (ES/PNET) is primarily a tumor of bone and soft tissues in the trunk or axial skeleton with most patients presenting in their second decade of life. The aggressive behavior of the tumor is evident by the advanced stage of presentation and frequent metastasis to various organs in 25% to 50% of patients [1], [2]. Ewing sarcoma/primitive neuroectodermal tumor is a rare primary tumor of the kidney, which can be mistaken for a variety of other round cell tumors primarily involving the kidney. They include blastemal predominant Wilms tumor (WT); monophasic synovial sarcoma (SS); lymphoma; clear cell sarcoma of the kidney (CCSK); small cell carcinoma; neuroblastoma; and desmoplastic small round cell tumor (DSRCT) [3]. Although these tumors share similar histology, each carries unique therapeutic and prognostic implications. The histomorphology of these tumors is variable, and the correct diagnosis is based on histologic and immunohistochemical features, complimented by cytogenetics and molecular analysis. A panel of immunohistochemical markers including MIC2, Fli-1, WT1, leukocyte common antigen (LCA), desmin, cytokeratin (CK), neuron specific enolase (NSE), and synaptophysin can be used to differentiate it from other small round cell tumors [4], [5]. With the advent of molecular markers that identify specific translocation in 95% of cases, the ES/PNET family of tumors is now defined by the presence of translocation between the EWS gene and members of the ETS family [6], [7], [8].