Tophat manual

Version 1. For convenience, the indels discovered during each run are also reported in BED files, similar to the junctions. We are very pleased to announce that you can now run TopHat and Cufflinks through Galaxy. The Galaxy project aims to make informatics tools accessible through the web, and allows you to experiment with parameter settings and create sophisticated analysis workflows easily.

Galaxy is developed by researchers at Emory University and Penn State in the Taylor and Nekrutenko labs, respectively. We are extremely grateful for the Galaxy team's work, and proud to have TopHat and Cufflinks offered through their platform. Several users pointed out that the recently released version 1. This has been corrected in the 1.

This is a strongly recommend fix release of TopHat. This bug affected users of unstranded RNA-Seq data as well as users of stranded reads, so 1. This release of TopHat adds support for strand-specific RNA-Seq alignment for reads produced by a number of strand-specific protocols. Please see the manual for details.

This release also supports variable-length reads. The build of 1. This has been corrected and the packages have been updated. Daehwan is a Ph. In response to user requests, Ben Langmead was kind enough to rebuild the Bowtie indexes for human and mouse from UCSC assembly fasta files. We recommend that users who need indexes other than human or mouse build them from UCSC fasta files.

Until recently, there was a bug that could cause TopHat to report no alignments or junctions with some Bowtie indexes including some indexes downloadable from this site. All users are strongly encouraged to upgrade to Bowtie 0. We're pleased to announce the release of a sister tool to TopHat, called Cufflinks. TopHat aligns your RNA-Seq reads; Cufflinks assembles those alignments into transcripts and also calculates isoform and gene level expression in your samples.

This TopHat release contains a number of stability improvements, fixes, and some substantial performance increases. The disk footprint is also reduced, though it's still large, and further reductions are coming in future releases. We advise all users to adopt Cufflinks to compute expression values. Cufflinks contains a sophisticated algorithm for this calculation, that is far more accurate than TopHat's method.

It is now ,, as intended. This release includes both fixes and new features. This upgrade requires Bowtie 0. Other changes including:. While the release of TopHat 1. This is mostly a fix release, but all users are encourage to upgrade, as some of the bugs fixed were fairly major. Other notable improvements include:. Reads longer than 50bp and paired end reads are substantially more powerful for finding splice junctions, and TopHat needed new algorithms to take advantage of them.

While this release should be considered a beta, and still contains bugs, it has been under development for several months and has been tested by several groups on both first- and second-generation RNA-Seq data in multiple organisms. Notable improvements include:. Our paper on discovering splice junctions has appeared at Bioinformatics. Our paper on discovering splice junctions has been accepted at Bioinformatics , and should appear soon.

TopHat 0. The code originally came from Rob's statistical alignment package FSA. The first public release of TopHat is now available for download. To use TopHat, you will need to install Bowtie and Maq. Both are open source and freely available under the Artistic license. When you install Bowtie, you should also install the Bowtie index for the genome in your RNA-Seq experiment, if one is available.

If there is no pre-built index for the organism you're interested in, you can follow the Bowtie manual's section on how to build one yourself. Because this is the first release, the manual is very limited. Only the basic options have been described. However, we will be updating it frequently, so please check back. If you find something unclear, or have questions about how TopHat works, please email Cole Trapnell.

We will be posting a list of frequently asked questions soon. In this release, TopHat does not consider mate pairing between reads. Use of mate pair information is our top development priority. Check back soon for a release with full paired-end support. It aligns RNA-Seq reads to mammalian-sized genomes using the ultra high-throughput short read aligner Bowtie , and then analyzes the mapping results to identify splice junctions between exons. New releases and related tools will be announced through the Bowtie mailing list.

Please use tophat. Please do not email technical questions to TopHat contributors directly. TopHat 2. This release implements a new algorithm for counting fusion-supporting read pairs that reduces the number of false-positive potential fusions.

This algorithm computes the inner distance between read pairs by first converting the pair positions to transcript coordinates using the transcript information in refGene. Pairs with small inner distance suggesting the pair could come from a plausible pair-end insert are counted as supporting evidence for the fusion. This includes reporting separate counts for the additional unpaired reads and making sure that the SAM flags in the output files reflect the paired or unpaired origin of the reads.

Added the possibility to run TopHat just for the purpose of preparing the transcriptome index files please see the manual entry for this special usage. Fixed a bug that could sometimes incorrectly rename the reads in the output alignments.

Save time with question packs and auto-grading, and run secure in-class tests or remote proctored exams. Plus, get insights to support class and individual progress. With the Weekly Course Report, reaching out to struggling students is only a click away. Every interaction—attendance, participation, assignments, tests—is automatically captured in the Top Hat Gradebook.

Get a view of each student's performance. Easily understand who's succeeding and who needs extra support. Engage your students with Top Hat and view grades, attendance and participation data in real time in the Gradebook.

Easily export to your LMS for the final tally. Use 15 different question types, like multiple choice, click-on-target and short answer. Securely administer in-class or remote quizzes, tests and exams on student devices. Let students ask questions, chat with each other and respond to your lecture with questions and emojis. Adopt or create a customizable interactive textbook that tracks student comprehension. Get students to sign into class using any device—in person or remotely.

Create auto-graded assignments tailored to your course. Performance, attendance and participation data are all easily exported to your LMS. We meet you where you are with one-on-one training, reliable technical support and instructional design experts to help you deliver your perfect course. Creating this Bowtie index can be time consuming and in many cases the same transcriptome data is being used for aligning multiple samples with TopHat. A transcriptome index and the associated data files the original GFF file can be thus reused for multiple TopHat runs with this option, so these files are only created for the first run with a given set of transcripts.

Then subsequent TopHat runs using the same --transcriptome-index option value will directly use the transcriptome data created in the first run no -G option needed after the first run.

Please note that starting with version 2. This is a special usage directing TopHat to only build the transcriptome index data files for the given annotation and then exit. Note: Only after the transcriptome files are built with one of the methods above, by a single TopHat process, it is safe to run multiple TopHat processes simultaneously making use of the same pre-built transcriptome index data.

The options below allow you validate your own indels with your RNA-Seq data. Supply TopHat with a list of insertions or deletions with respect to the reference.

Indels are specified one per line, in a tab-delimited format, identical to that of junctions. Records are formatted as follows:. For example: chr1 For instance: chr1 CA.. The tophat script produces a number of files in the directory in which it was invoked.

Most of these files are internal, intermediate files that are generated for use within the pipeline. The output files you will likely want to look at are:. New releases and related tools will be announced through the Bowtie mailing list. Please use tophat. Please do not email technical questions to TopHat contributors directly. Manual What is TopHat? Prerequisites Using TopHat. Please note: TopHat has a number of parameters and options, and their default values are tuned for processing mammalian RNA-Seq reads.

If you would like to use TopHat for another class of organism, we recommend setting some of the parameters with more strict, conservative values than their defaults. Usually, setting the maximum intron size to 4 or 5 Kb is sufficient to discover most junctions while keeping the number of false positives low. Administrator: Daehwan Kim. Design by David Herreman. Source code. The basename of the genome index to be searched. The basename is the name of any of the index files up to but not including the first period.

Final read alignments having more than these many mismatches are discarded. The default is 2. Final read alignments having more than these many total length of gaps are discarded. Final read alignments having more than these many edit distance are discarded. Some of the reads spanning multiple exons may be mapped incorrectly as a contiguous alignment to the genome even though the correct alignment should be a spliced one - this can happen in the presence of processed pseudogenes that are rarely if at all transcribed or expressed.

This option can direct TopHat to re-align reads for which the edit distance of an alignment obtained in a previous mapping step is above or equal to this option value. If you set this option to 0 , TopHat will map every read in all the mapping steps transcriptome if you provided gene annotations, genome, and finally splice variants detected by TopHat , reporting the best possible alignment found in any of these mapping steps.

This may greatly increase the mapping accuracy at the expense of an increase in running time. The default value for this option is set such that TopHat will not try to realign reads already mapped in earlier steps. Uses Bowtie1 instead of Bowtie2. If you use colorspace reads, you need to use this option as Bowtie2 does not support colorspace reads. Sets the name of the directory in which TopHat will write all of its output. The default is ".

This is the expected mean inner distance between mate pairs. For, example, for paired end runs with fragments selected at bp, where each end is 50bp, you should set -r to be The default is 50bp.

The standard deviation for the distribution on inner distances between mate pairs. The default is 20bp. The "anchor length". TopHat 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.

This must be at least 3 and the default is 8. The maximum number of mismatches that may appear in the "anchor" region of a spliced alignment. The default is 0. The minimum intron length. The default is