Determining the upstream and downstream genes in fusion events. (a) A fragment of paired-end sequencing is shown with its complementary fragment. Paired-end reads (reads 'a' and 'b') are shown with their sequencing direction (from 5' to 3', noted by arrows on reads). Read 'a' is generated from the fragment itself, while read 'b' is from the complementary fragment. The sequencing orientation is from bilateral edges to the middle of the fragment, so the paired-end reads are generated head-to-head. (b,c) Different classifications of span-read (read 'a' and 'b') support different upstream and downstream genes. The gene aligned by reads in the plus orientation must be the upstream gene. In (b), read 'a' aligns to Gene A in a plus orientation. Based on the paired-end sequencing shown in (a), Gene A must be the upstream gene and Gene B must be the downstream gene. In (c), read 'b' aligns to Gene B in a plus orientation. So, Gene B is an upstream gene and Gene A is a downstream gene.