June 1, 2021  |  

Scalability and reliability improvements to the Iso-Seq analysis pipeline enables higher throughput sequencing of full-length cancer transcripts

The characterization of gene expression profiles via transcriptome sequencing has proven to be an important tool for characterizing how genomic rearrangements in cancer affect the biological pathways involved in cancer progression and treatment response. More recently, better resolution of transcript isoforms has shown that this additional level of information may be useful in stratifying patients into cancer subtypes with different outcomes and responses to treatment.1 The Iso-Seq protocol developed at PacBio is uniquely able to deliver full-length, high-quality cDNA sequences, allowing the unambiguous determination of splice variants, identifying potential biomarkers and yielding new insights into gene fusion events. Recent improvements to the Iso-Seq bioinformatics pipeline increases the speed and scalability of data analysis while boosting the reliability of isoform detection and cross-platform usability. Here we report evaluation of Sequel Iso-Seq runs of human UHRR samples with spiked-in synthetic RNA controls and show that the new pipeline is more CPU efficient and recovers more human and synthetic isoforms while reducing the number of false positives. We also share the results of sequencing the well-characterized HCC-1954 breast cancer and normal breast cell lines, which will be made publicly available. Combined with the recent simplification of the Iso-Seq sample preparation2, the new analysis pipeline completes a streamlined workflow for revealing the most comprehensive picture of transcriptomes at the throughput needed to characterize cancer samples.

June 1, 2021  |  

The role of androgen receptor variant AR-V9 in prostate cancer

The expression of androgen receptor (AR) variants is a frequent, yet poorly-understood mechanism of clinical resistance to AR-targeted therapy for castration-resistant prostate cancer (CRPC). Among the multiple AR variants expressed in CRPC, AR-V7 is considered the most clinically-relevant AR variant due to broad expression in CRPC, correlations of AR-V7 expression with clinical resistance, and growth inhibition when AR-V7 is knocked down in CRPC models. Therefore, efforts are under way to develop strategies for monitoring and inhibiting AR-V7 in castration-resistant prostate cancer (CRPC). The aim of this study was to understand whether other AR variants are co-expressed with AR-V7 and promote resistance to AR-targeted therapies. To test this, we utilized RNA-seq to characterize AR expression in CRPC models. RNA-seq revealed the frequent coexpression of AR-V9 and AR-V7 in multiple CRPC models and metastases. Furthermore, long-read single-molecule real-time (SMRT) sequencing of AR isoforms revealed that AR-V7 and AR-V9 shared a common 3’terminal cryptic exon. To test this, we knocked down AR-V7 in prostate cancer cell lines and confirmed that AR-V9 mRNA and protein expression were also impacted. In reporter assays with AR-responsive promoters, AR-V9 functioned as a constitutive activator of androgen/AR signaling. Similarly, infection of AR-V9 lentiviral construct in LNCaP cells induced androgen-independent cell proliferation. In conclusion, these data implicate co-expression of AR-V9 with AR-V7 as an important component of constitutive AR signaling and therapeutic resistance in CRPC.

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