Top 10 overgrowth mods12/16/2023 The reaction followed the NEBNext Single Cell/Low Input protocol (catalog number E6421S New England Biolabs, Ipswich, MA). The HBR RNA sample spiked with 2% SIRV-Set 4 will be referred to as HBR/SIRV for the remainder of the text. The exemplar case studies demonstrate that Iso-Seq and PB_FLIP discover novel expressed fusion partners, resolve complex intragenic alterations, and discriminate between allele-specific expression profiles.įirst-strand cDNA synthesis used 300 ng of total tumor or HBR RNA (Ambion, Austin, TX catalog number AM7962) spiked with a 2% SIRV-Set 4 synthetic RNA mix (Spike-In RNA Variants Lexogen, Vienna, Austria catalog number 141). This study describes the utility of Iso-Seq and PB_FLIP analysis in improving deconvolution of complex structural variants and isoform detection within an institutional pediatric and adolescent/young adult translational cancer research cohort. In addition, sequencing of a commercial reference (Spike-In RNA Variants) with known isoform complexity was performed and demonstrated high recall of the Iso-Seq and PB_FLIP workflow to benchmark our protocol and analysis performance. ![]() The Pacific Biosciences Fusion and Long Isoform Pipeline (PB_FLIP) incorporates a suite of RNA-sequencing software analysis tools and scripts to identify expressed fusion partners and isoforms. ![]() For example, the Pacific Biosciences long-read RNA-sequencing (Iso-Seq) transcriptome protocol provides full-length isoform characterization, discernment of allelic phasing, and isoform discovery, and identifies expressed fusion partners. However, given the frequent occurrence of structural variation in cancer, molecular profiling using long-read sequencing improves the resolution of such events. Genomic profiling using short-read sequencing has utility in detecting disease-associated variation in both DNA and RNA.
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