## Alignment / TopHat2 for paired-end reads

### Description

This tool aligns Illumina paired-end RNA-seq reads to publicly available genomes. You need to supply the reads in two fastq files containing the reads in the same order. You can also supply a GTF file, if you would like TopHat to use existing splice site information. If you would like us to add new reference genomes to Chipster, please contact us.

### Parameters

- Genome (human hg19, mouse mm10, rat rn4) [mouse mm9]
- Expected inner distance between mate pairs (1-1000) [200]
- Standard deviation for the inner distances between mate pairs (Int) [20]
- Minimum anchor length (3-1000) [8]
- Maximum number of mismatches allowed in the anchor (0, 1, 2) [0]
- Minimum intron length (10-1000) [70]
- How many hits is a read allowed to have (1-1000000) [20]
- Use annotation GTF (yes, no) [yes]
- When GTF file is used, ignore novel junctions (yes, no) [yes]

### Details

TopHat2 first identifies potential exons by mapping the reads to the genome using the Bowtie aligner. Using this initial mapping, it builds a database of possible splice junctions, and then maps the reads against these junctions to confirm them. As many exons are shorter than reads, TopHat2 splits the reads into smaller segments, which are then mapped independently. The segment alignments are "glued" back together in a final step of the program to produce end-to-end read alignments. TopHat generates its database of possible splice junctions from three sources of evidence:

- Pairings of "coverage islands", which are distinct regions of piled up reads in the initial mapping. Neighboring islands are often spliced together in the transcriptome, so TopHat2 looks for ways to join these with an intron.
- When reads in a pair come from different exons of a transcript, they will generally map far apart in the genome coordinate space. When this happens, TopHat2 tries to "close" the gap between them by looking for subsequences of the genomic interval between mates with a total length about equal to the expected distance between mates. The "introns" in this subsequence are added to the database.
- Two segments from the same read are mapped far apart, or when an internal segment fails to map.

You have to give the expected mean inner distance between mate pairs. For example, if your fragment size is 300 bp and read length is 50 bp, the inner distance is 200.

The "anchor length" means that TopHat2 will report junctions spanned by reads with at least this many bases on each side of the junction. Note that individual spliced alignments may span a junction with fewer than this many bases on one side. However, every junction involved in spliced alignments is supported by at least one read with this many bases on each side. By default no mismatches are allowed in the anchor, but you can change this.

TopHat2 will ignore donor-acceptor pairs which are closer than the minimum intron length or further than the maximum intron length apart. With long (>=75bp) reads, "GT-AG", "GC-AG" and "AT-AC" introns can be found ab initio. With shorter reads, TopHat only reports alignments across "GT-AG" introns.

TopHat can optionally use existing gene model annotations (splice sites). If GTF file is supplied (by the user or available from the server), TopHat will use the exon records in this file to build a set of known splice site junctions for each gene, and it will attempt to align reads to these junctions even if they would not normally be covered by the initial mapping. If the GTF file is used, the user can specify if TopHat should look for reads across the known splice junctions only.

After running TopHat2, Chipster indexes the BAM file using the SAMtools package. This way the results are ready to be visualized in the genome browser.

### Output

This tool returns a BAM file containing the alignment, and an index file (.bai) for it. In addition it produces the following BED files:

- junctions.bed. Each junction consists of two connected BED blocks, where each block is as long as the maximal overhang of any read spanning the junction. The score is the number of alignments spanning the junction.
- insertions.bed. Insertions reported by TopHat. chromLeft refers to the last genomic base before the insertion.
- deletions.bed. Deletions reported by TopHat. chromLeft refers to the first genomic base of the deletion.

### Reference

This tool is based on the TopHat package. Please cite the following article:

Trapnell C, Pachter L, Salzberg SL. TopHat: discovering splice junctions with RNA-Seq. Bioinformatics (2009) 25 (9): 1105-1111.