TopHat

exon1
may be treated as intron exon2 exon3
exon1
exon3
TopHat
• a fast splice junction mapper for RNA-Seq reads. It aligns RNA-Seq reads to mammaliansized genomes using the ultra highthroughput short read aligner Bowtie, and then analyzes the mapping results to identify splice junctions between exons. • TopHat cuts each read into multiple segments whose size is 25b by default
• transcript has very low sequencing coverage • Junctions spanning very long introns or introns with non-canonical donor and acceptor sites (such as GC–AG introns) • single-island junctions in islands with a low normalized depth of coverage • Based on the mapping of Bowtie • multireads, • Missing extrons
• single-island junctions tend to fall within islands with high D • TopHat looks for junctions contained in islands with D≥300, this parameter can be changed by the user
• the pipeline maps all reads to the reference genome using Bowtie-- putative exons
• All reads that do not map to the genome are set aside as ‘initially unmapped reads’, or IUM reads
• when two segments from the same read (for reads of at least 45bp) are mapped at a certain distance on the same genomic sequence or when an internal segment fails to map - again suggesting that such reads are spanning multiple exons. With this approach, "GT-AG", "GC-AG" and "AT-AC" introns will be found ab iniions with RNA-Seq
• expressed sequence tags (ESTs) • RNA-Seq: 1. sample the mRNAwith fewer biases 2. generates far more data per experiment 3. a direct measure of the level of gene expression.
2k-mer
2k-mer
S-2k+1
• Neighboring islands are often spliced together in the transcriptome, so TopHat looks for ways to join these with an intron • for short reads (< 45bp) and with a small number of reads (<= 10 million). • only report alignments across "GT-AG" introns
• enumerates all canonical donor and acceptor sites within the island sequences
• considers all pairings of these sites that could form canonical (GT–AG) introns between neighboring islands
• Short read sequencing machines can currently produce reads 100bp or longer but many exons are shorter than this so they would be missed in the initial mapping • TopHat ----splitting all input reads into smaller segments which are then mapped independently • The segment alignments are put back together in a final step of the program to produce the endto-end read alignments.