TopHat2 is an advanced spliced aligner for RNA-seq data, designed to handle various lengths of reads and variable-length indels. It can align reads across fusion breaks and identify novel splice sites, producing accurate alignments even in repetitive genomes or the presence of pseudogenes. TopHat2 combines de novo spliced alignment with direct mapping to known transcripts, enhancing sensitivity and accuracy. The software uses a two-step procedure to detect potential splice sites and align multi-exon-spanning reads. It also incorporates algorithms to handle pseudogenes and structural variations, such as insertions, deletions, and translocations. TopHat2 is available at http://ccb.jhu.edu/software/tophat. The paper evaluates TopHat2's performance through simulations and real data, demonstrating its superior accuracy compared to other aligners like GSNAP, RUM, STAR, and MapSplice, especially in handling short-anchored reads and indels. TopHat2's realignment capability further improves alignment accuracy, particularly for known splice sites. The software is optimized for long paired-end reads and is efficient in terms of computational resources.TopHat2 is an advanced spliced aligner for RNA-seq data, designed to handle various lengths of reads and variable-length indels. It can align reads across fusion breaks and identify novel splice sites, producing accurate alignments even in repetitive genomes or the presence of pseudogenes. TopHat2 combines de novo spliced alignment with direct mapping to known transcripts, enhancing sensitivity and accuracy. The software uses a two-step procedure to detect potential splice sites and align multi-exon-spanning reads. It also incorporates algorithms to handle pseudogenes and structural variations, such as insertions, deletions, and translocations. TopHat2 is available at http://ccb.jhu.edu/software/tophat. The paper evaluates TopHat2's performance through simulations and real data, demonstrating its superior accuracy compared to other aligners like GSNAP, RUM, STAR, and MapSplice, especially in handling short-anchored reads and indels. TopHat2's realignment capability further improves alignment accuracy, particularly for known splice sites. The software is optimized for long paired-end reads and is efficient in terms of computational resources.