Validation of branchpoint annotations. (A) Schematic indicating incorporation of mismatch nucleotide (gray X) within cDNA (blue/orange bar) by reverse transcriptase when traversing the 2′ to 5′ branch junction. (B) UCSC Genome Browser view of branchpoints within EZH2 gene (lower panel) showing capture of sequences at intron ends (histogram) and sequenced read alignments that indicate branchpoints (red/blue bar). RT-PCR using outward facing primers (blue) generates an amplicon that validates branchpoint annotations (blue/orange upper panel). Zoom of chromatograph (top left) shows sequence mismatch error (T) at the branchpoint nucleotide (boxed). (C) Histogram indicates rate of mismatch errors at the branchpoint nucleotide in sequenced reads that align across the lariat junction (center). (D) Summary of branchpoint annotations with varying levels of support: split/inverted alignment, exact match across lariat junction, match with mismatch, or insertion/deletion errors at branchpoints. At each level of support, the number of identified branchpoints, nucleotide composition of the branchpoint, and fraction of GENCODE introns with an annotated branchpoint are indicated.
