TopHat

A spliced read mapper for RNA-Seq

       

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    Questions about TopHat should be sent to tophat.cufflinks@gmail.com. Please do not email technical questions to TopHat contributors directly.

Releases

Related Tools

  • Cufflinks: Isoform assembly and quantitation for RNA-Seq
  • Bowtie: Ultrafast short read alignment
  • TopHat-Fusion: An algorithm for Discovery of Novel Fusion Transcripts
  • CummeRbund: Visualization of RNA-Seq differential analysis

Pre-built indexes
H. sapiens, UCSC hg18 2.7 GB
 colorspace: full
H. sapiens, UCSC hg19 2.7 GB
 colorspace: full
M. musculus, UCSC mm9 2.4 GB
 colorspace: full

All indexes are for assemblies, not contigs. Unplaced or unlocalized sequences and alternate haplotype assemblies are excluded.

Some unzip programs cannot handle archives >2 GB. If you have problems downloading or unzipping a >2 GB index, try downloading in two parts.

Check .zip file integrity with MD5s.

Pre-built indexes are compatible with Bowtie versions 0.9.8 and later. For older indexes, please contact us.

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Contributors

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Manual


What is TopHat?


TopHat is a program that aligns RNA-Seq reads to a genome in order to identify exon-exon splice junctions. It is built on the ultrafast short read mapping program Bowtie. TopHat runs on Linux and OS X.


What types of reads can I use TopHat with?


TopHat was designed to work with reads produced by the Illumina Genome Analyzer, although users have been successful in using TopHat with reads from other technologies. In TopHat 1.1.0, we began supporting Applied Biosystems' Colorspace format. The software is optimized for reads 75bp or longer.

Mixing paired- and single- end reads together is not supported.


How does TopHat find junctions?


TopHat finds splice junctions without a reference annotation. By first mapping RNA-Seq reads to the genome, TopHat identifies potential exons, since many RNA-Seq reads will contiguously align to the genome. Using this initial mapping information, TopHat builds a database of possible splice junctions, and then maps the reads against these junctions to confirm them.

Short read sequencing machines can currently produce reads 100bp or longer, but many exons are shorter than this, and so would be missed in the initial mapping. TopHat solves this problem by splitting all input reads into smaller segments, and then mapping them independently. The segment alignments are "glued" back together in a final step of the program to produce the end-to-end read alignments.

TopHat generates its database of possible splice junctions from three sources of evidence. The first source is 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 TopHat looks for ways to join these with an intron. The second source is only used when TopHat is run with paired end reads. When reads in a pair come from different exons of a transcript, they will generally be mapped far apart in the genome coordinate space. When this happens, TopHat 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. The third, and strongest, source of evidence for a splice junction is when two segments from the same read are mapped far apart, or when an internal segment fails to map. With long (>=75bp) reads, "GT-AG", "GC-AG" and "AT-AC" introns will be found ab initio. With shorter reads, TopHat only reports alignments across "GT-AG" introns


Prerequisites


To use TopHat, you will need the following programs in your PATH:

  • bowtie2 and bowtie2-align (or bowtie)
  • bowtie2-inspect (or bowtie-inspect)
  • bowtie2-build (or bowtie-build)
  • samtools

Because TopHat outputs and handles alignments in BAM format, you will need to download and install the SAM tools. You may want to take a look at the Getting started guide for more detailed installation instructions, including installation of SAM tools and Boost.

You will also need Python version 2.4 or higher.


Obtaining and installing TopHat


You can download the latest source release and precompiled binaries for Linux and Mac OSX here. See the Getting started guide for detailed instructions about installing TopHat from the binary package or building TopHat and its dependencies from source.

To install TopHat from source package, unpack the tarball and change directory to the package directory as follows:

tar zxvf tophat-2.0.0.tar.gz
cd tophat-2.0.0/

Configure the package, specifying the install path and the library dependencies as needed (see the  Getting started guide for details):

./configure --prefix=<install_prefix> --with-boost=<boost_install_prefix> --with-bam=<samtools_install_prefix>

Finally, build and install TopHat:

make
make install

As detailed in the Getting started guide, if you want to install TopHat 2 without overwriting a previous version of TopHat already installed on your system you should specify a new, separate <install_prefix> for the ./configure command above, and after the 'make install' step just copy the tophat2 script from <install_prefix>/bin to a directory that is in your shell's PATH, so you can invoke this new version of TopHat with the command 'tophat2'.

Below you will find a detailed list of command-line options you can use to control TopHat. Beginning users should take a look at the Getting started guide for a tutorial on installing and running TopHat and its prerequisites.

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.

Using TopHat


The following is a detailed description of the options used to control the tophat script:


Usage: tophat [options]* <index_base> <reads1_1[,...,readsN_1]> [reads1_2,...readsN_2]
When running TopHat with paired ends, it is critical that the *_1 files an the *_2 files appear in separate comma separated lists, and that the order of the files in the two lists is the same.

NOTE: TopHat can align reads that are up to 1024 bp, and it handles paired end reads, but we do not recommend mixing several "types" of reads in the same TopHat run. For example, mixing 100bp single end reads and 2x27bp paired ends into the same TopHat run will give bad results. If you'd like to combine results from several "flavors" of RNA-Seq reads, you can run first with one of your sets, and feed the junctions produced by that run into future TopHat runs as externally supplied junctions with the -j option (see below)

Arguments:
<ebwt_base> The basename of the index to be searched. The basename is the name of any of the five index files up to but not including the first period. bowtie first looks in the current directory for the index files, then looks in the indexes subdirectory under the directory where the currently-running bowtie executable is located, then looks in the directory specified in the BOWTIE_INDEXES environment variable.
<reads1_1[,...,readsN_1]> A comma-separated list of files containing reads in FASTQ or FASTA format. When running TopHat with paired-end reads, this should be the *_1 ("left") set of files.
<[reads1_2,...readsN_2]> A comma-separated list of files containing reads in FASTA or FASTA format. Only used when running TopHat with paired end reads, and contains the *_2 ("right") set of files. The *_2 files MUST appear in the same order as the *_1 files.
Options:
-h/--help Prints the help message and exits
-v/--version Prints the TopHat version number and exits
--bowtie1 Uses Bowtie1 instead of Bowtie2. If you use colorspace reads, you need to use this option as Bowtie2 does not support colorspace reads.
-o/--output-dir <string> Sets the name of the directory in which TopHat will write all of its output. The default is "./tophat_out".
-r/--mate-inner-dist <int> This is the expected (mean) inner distance between mate pairs. For, example, for paired end runs with fragments selected at 300bp, where each end is 50bp, you should set -r to be 200. There is no default, and this parameter is required for paired end runs.
--mate-std-dev <int> The standard deviation for the distribution on inner distances between mate pairs. The default is 20bp.
-a/--min-anchor-length <int> 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.
-m/--splice-mismatches <int> The maximum number of mismatches that may appear in the "anchor" region of a spliced alignment. The default is 0.
-i/--min-intron-length <int> The minimum intron length. TopHat will ignore donor/acceptor pairs closer than this many bases apart. The default is 70.
-I/--max-intron-length <int> The maximum intron length. When searching for junctions ab initio, TopHat will ignore donor/acceptor pairs farther than this many bases apart, except when such a pair is supported by a split segment alignment of a long read. The default is 500000.
--max-insertion-length <int> The maximum insertion length. The default is 3.
--max-deletion-length <int> The maximum deletion length. The default is 3.
--solexa-quals Use the Solexa scale for quality values in FASTQ files.
--solexa1.3-quals As of the Illumina GA pipeline version 1.3, quality scores are encoded in Phred-scaled base-64. Use this option for FASTQ files from pipeline 1.3 or later.
-Q/--quals Separate quality value files - colorspace read files (CSFASTA) come with separate qual files.
--integer-quals Quality values are space-delimited integer values, this becomes default when you specify -C/--color.
-C/--color Colorspace reads, note that it uses a colorspace bowtie index and requires Bowtie 0.12.6 or higher.
Common usage: tophat --color --quals [other options]* <colorspace_index_base> <reads1_1[,...,readsN_1]> [reads1_2,...readsN_2] <quals1_1[,...,qualsN_1]> [quals1_2,...qualsN_2]
-p/--num-threads <int> Use this many threads to align reads. The default is 1.
-g/--max-multihits <int> Instructs TopHat to allow up to this many alignments to the reference for a given read, and suppresses all alignments for reads with more than this many alignments. The default is 20 for read mapping.
--report-secondary-hits Without the option, TopHat will report best or primary alignments based on alignment scores (AS). If you want to output additional or secondary alignments, use the option, which will report up to 20 alignments (the default number 20 can be changed using -g/--max-multihits option above).
--report-discordant-pair-alignments This option will allow mate pairs to map to different chromosomes, distant places on the same chromosome, or on the same strand.
--no-coverage-search Disables the coverage based search for junctions.
--coverage-search Enables the coverage based search for junctions. Use when coverage search is disabled by default (such as for reads 75bp or longer), for maximum sensitivity.
--microexon-search With this option, the pipeline will attempt to find alignments incident to microexons. Works only for reads 50bp or longer.
--library-type TopHat will treat the reads as strand specific. Every read alignment will have an XS attribute tag. Consider supplying library type options below to select the correct RNA-seq protocol.
Library TypeExamplesDescription
fr-unstrandedStandard IlluminaReads from the left-most end of the fragment (in transcript coordinates) map to the transcript strand, and the right-most end maps to the opposite strand.
fr-firststranddUTP, NSR, NNSRSame as above except we enforce the rule that the right-most end of the fragment (in transcript coordinates) is the first sequenced (or only sequenced for single-end reads). Equivalently, it is assumed that only the strand generated during first strand synthesis is sequenced.
fr-secondstrandLigation, Standard SOLiDSame as above except we enforce the rule that the left-most end of the fragment (in transcript coordinates) is the first sequenced (or only sequenced for single-end reads). Equivalently, it is assumed that only the strand generated during second strand synthesis is sequenced.
Advanced Options:
-n/--transcriptome-mismatches Maximum number of mismatches allowed when reads are aligned to the transcriptome. The default is 2. When Bowtie2 is used, this number is also used to decide whether or not to further re-align some of the transcriptome-mapped reads to the genome. If the alignment score of the best alignment among multiple candidates for a read is lower than "bowtie2-min-score", which is internally defined as (max_penalty - 1) * max_mismatches, then the reads will be kept for re-alignment through the rest of the pipeline.  You can specify max_penalty via "--b2-mp" option.
--genome-read-mismatches When whole reads are first mapped on the genome, this many mismatches in each read alignment are allowed. The default is 2. This number is also used to decide whether to further re-align some of the reads (by splitting them into segments) with a similar scoring threshold scheme as described for the --transcriptome-mismatches option above.
--read-mismatches Final read alignments having more than these many mismatches are discarded. The default is 2.
--bowtie-n TopHat uses "-v" in Bowtie for initial read mapping (the default), but with this option, "-n" is used instead. Read segments are always mapped using "-v" option.
--segment-mismatches Read segments are mapped independently, allowing up to this many mismatches in each segment alignment. The default is 2.
--segment-length Each read is cut up into segments, each at least this long. These segments are mapped independently. The default is 25.
--min-coverage-intron The minimum intron length that may be found during coverage search. The default is 50.
--max-coverage-intron The maximum intron length that may be found during coverage search. The default is 20000.
--min-segment-intron The minimum intron length that may be found during split-segment search. The default is 50.
--max-segment-intron The maximum intron length that may be found during split-segment search. The default is 500000.
--keep-tmp Causes TopHat to preserve its intermediate files produced during the run (mostly useful for debugging). The default is to delete these temporary files.
--keep-fasta-odrer In order to sort alignments in the same order in the genome fasta file, the option can be used. But this option will make the output SAM/BAM file incompatible with those from the previous versions of TopHat (1.4.1 or lower).
--no-sort-bam Output BAM is not coordinate-sorted.
--no-convert-bam Do not convert to bam format. Output is <output_dir>/accepted_hit.sam. Implies --no-sort-bam.
-z/--zpacker Manually specify the program used for compression of temporary files; default is gzip; use -z0 to disable compression altogether. Any program that is option-compatible with gzip can be used (e.g. bzip2, pigz, pbzip2).

Bowtie 2 specific options:

Bowtie 2 provides many options so that users can have more flexibility as to how reads are mapped. TopHat 2 allows users to pass many of these options to Bowtie 2 by preceding the Bowtie 2 option name with the --b2- prefix.  Please refer to the Bowtie2 website for detailed information.

Preset options in --end-to-end mode  (local alignment is not used in TopHat2):
Tophat 2 option:
 Corresponding Bowtie 2 option:
--b2-very-fast --very-fast
--b2-fast --fast
--b2-sensitive --sensitive
--b2-very-sensitive --very-sensitive
Alignment options:
--b2-N The default is 0.
--b2-L The default is 20.
--b2-i The default is S,1,1.25.
--b2-n-ceil The default is L,0,0.15.
--b2-gbar The default is 4.
Scoring options:
--b2-mp The default is 6,2.
--b2-np The default is 1.
--b2-rdg The default is 5,3.
--b2-rfg The default is 5,3.
--b2-score-min The default is L,-0.6,-0.6.
Effort options:
--b2-D The default is 15.
--b2-R The default is 2.
Fusion mapping options:

Reads can be aligned to potential fusion transcripts if the --fusion-search option is specified. The fusion alignments are reported in SAM format using custom fields XF and XP (see the output format) and some additional information about fusions will be reported (see fusions.out). Once mapping is done, you can run tophat-fusion-post to filter out fusion transcripts (see the TopHat-Fusion website for more details).

--fusion-search Turn on fusion mapping
--fusion-anchor-length A "supporting" read must map to both sides of a fusion by at least this many bases. The default is 20.
--fusion-min-dist For intra-chromosomal fusions, TopHat-Fusion tries to find fusions separated by at least this distance. The default is 10000000.
--fusion-read-mismatches Reads support fusions if they map across fusion with at most this many mismatches. The default is 2.
--fusion-multireads Reads that map to more than this many places will be ignored. It may be possible that a fusion is supported by reads (or pairs) that map to multiple places. The default is 2.
--fusion-multipairs Pairs that map to more than this many places will be ignored. The default is 2.
--fusion-ignore-chromosomes Ignore some chromosomes such as chrM when detecting fusion break points. Please check the correct names for chromosomes, that is, mitochondrial DNA is represented as chrM or M depending on the annotation you use.
Supplying your own transcript annotation data:

The options below allow you validate your own list of known transcripts or junctions with your RNA-Seq data. Note that the chromosome names in the files provided with the options below must match the names in the Bowtie index. These names are case-senstitive.
-j/--raw-juncs <.juncs file>

Supply TopHat with a list of raw junctions. Junctions are specified one per line, in a tab-delimited format. Records look like:


<chrom> <left> <right> <+/->

left and right are zero-based coordinates, and specify the last character of the left sequenced to be spliced to the first character of the right sequence, inclusive. That is, the last and the first positions of the flanking exons. Users can convert junctions.bed (one of the TopHat outputs) to this format using bed_to_juncs < junctions.bed > new_list.juncs where bed_to_juncs can be found under the same folder as tophat

--no-novel-juncs Only look for reads across junctions indicated in the supplied GFF or junctions file. (ignored without -G/-j)
-G/--GTF <GTF/GFF3 file>

Supply TopHat with a set of gene model annotations and/or known transcripts, as a GTF 2.2 or GFF3 formatted file. If this option is provided, TopHat will first extract the transcript sequences and use Bowtie to align reads to this virtual transcriptome first. Only the reads that do not fully map to the transcriptome will then be mapped on the genome. The reads that did map on the transcriptome will be converted to genomic mappings (spliced as needed) and merged with the novel mappings and junctions in the final tophat output.

Please note that the values in the first column of the provided GTF/GFF file (column which indicates the chromosome or contig on which the feature is located), must match the name of the reference sequence in the Bowtie index you are using with TopHat. You can get a list of the sequence names in a Bowtie index by typing:


bowtie-inspect --names your_index

So before using a known annotation file with this option please make sure that the 1st column in the annotation file uses the exact same chromosome/contig names (case sensitive) as shown by the bowtie-inspect command above.
--transcriptome-index <dir/prefix>

When providing TopHat with a known transcript file (-G/--GTF option above), a transcriptome sequence file is built and a Bowtie index has to be created for it in order to align the reads to the known transcripts. 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. If multiple TopHat runs are planned with the same transcriptome data, TopHat should be first run with the -G option and with the --transcriptome-index option pointing to a directory and a name prefix which will indicate where the transcriptome data files will be stored. 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 for subsequent runs).

For example the first TopHat run could look like this:
tophat -o out_sample1 -G known_genes.gtf \
--transcriptome-index=transcriptome_data/known \
hg19 sample1_1.fq.z
In this example the first run will create the transcriptome_data directory if it doesn't exist, and files known.fa, known.gff and known.*ebwt (Bowtie index files) will be generated in that directory. Then for subsequent runs with the same genome and known transcripts but different reads (e.g. sample2_2.fq.z etc.), TopHat will no longer spend time building the transcriptome index because it can directly use the previously built transcriptome index, so the -G option can be even discarded for subsequent runs:
tophat -o out_sample2 \
--transcriptome-index=transcriptome_data/known \
hg19 sample2_1.fq.z
(The following options in this section are only used when the transcriptome search was activated with -G/--GTF or --transcriptome-index)
-T/--transcriptome-only Only align the reads to the transcriptome and report only those mappings as genomic mappings.
-x/--transcriptome-max-hits Maximum number of mappings allowed for a read, when aligned to the transcriptome (any reads found with more then this number of mappings will be discarded).
-M/--prefilter-multihits When mapping reads on the transcriptome, some repetitive or low complexity reads that would be discarded in the context of the genome may appear to align to the transcript sequences and thus may end up reported as mapped to those genes only. This option directs TopHat to first align the reads to the whole genome in order to determine and exclude such multi-mapped reads (according to the value of the -g/--max-multihits option).
Supplying your own insertions/deletions:

The options below allow you validate your own indels with your RNA-Seq data. Note that the chromosome names in the files provided with the options below must match the names in the Bowtie index. These names are case-senstitive.
--insertions/--deletions <.juncs file>

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 look like:


<chrom> <left> <right> <+/->

left and right are zero-based coordinates, and specify the last character of the left sequenced to be spliced to the first character of the right sequence, inclusive.

--no-novel-indels Only look for reads across indels in the supplied indel file, or disable indel detection when no file has been provided.

TopHat Output


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:


  1. accepted_hits.bam. A list of read alignments in SAM format. SAM is a compact short read alignment format that is increasingly being adopted. The formal specification is here.
  2. junctions.bed. A UCSC BED track of junctions reported by TopHat. 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.
  3. insertions.bed and deletions.bed. UCSC BED tracks of insertions and deletions reported by TopHat.
    Insertions.bed - chromLeft refers to the last genomic base before the insertion.
    Deletions.bed - chromLeft refers to the first genomic base of the deletion.