Fig. 1

PacBio Full-Length Isoform Concatemer Sequencing (PB_FLIC-Seq) overview. 1) First-strand cDNA generation using oligo(dT) priming. 2) Biotin addition via cDNA amplification for pending template switching oligo (TSO) artifact removal. 3) Bead based capture to remove TSO artifacts. 4) cDNA size-selection prior to concatemer formation. 5) PCR using primers containing known reverse-complement barcodes with uracil incorporation ("dU”), followed by USER digestion and ligation to form the concatemer array. 6) Samples undergo PacBio SMRTbell prep, sequencing, and PacBio primary analysis including SKERA for read splitting. Deconcatenated reads are used as input into the PacBio Fusion and Long Isoform Pipeline “PB_FLIP” [17] for isoform characterization and gene fusion detection