Tophat
2013-12-23 17:12
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What is 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. |
Using TopHat
Usage: tophat [options]* <genome_index_base> <reads1_1[,...,readsN_1]> [reads1_2,...readsN_2] When running TopHat with paired reads it is critical that the *_1 files an the *_2 files appear in separate comma-delimited lists, and that the order of the files in the two lists is the same.
TopHat allows the use of additional unpaired reads to be provided after the
paired reads. These unpaired reads can be either given at the end of the paired read files on one side (as reads that can no longer be paired with reads from the other side), or they can be given in separate file(s) which are appended (comma delimited) to
the list of paired input files on either side e.g.:
tophat [options]* <genome_index_base> PE_reads_1.fq.gz,SE_reads.fa PE_reads_2.fq.gz
‐ or ‐
tophat [options]* <genome_index_base> PE_reads_1.fq.gz PE_reads_2.fq.gz,SE_reads.fa
Starting with version 2.0.10 TopHat accepts mixed input file formats (FASTA/FASTQ).
NOTE: TopHat can align reads that are up to 1024 bp long, and it handles paired-end reads and unpaired reads at once, but we do not recommend mixing different types of reads in the same TopHat run. For example, mixing
100bp single end reads and 2x27bp paired reads in the same TopHat run may give sub-optimal results. If you'd like to combine results from data sets with different types of RNA-Seq reads, you can follow a protocol like this:
run TopHat on the first set of reads, with the appropriate parameters for this data set
use bed_to_juncs to convert the junctions.bed file obtained in this first run to a junction file usable by Tophat's -j option
run Tophat on the 2nd set of reads using the -j option to supply the junctions file produced by bed_to_juncs in the previous step
The following is a detailed description of the options used to control the TopHat script.
Arguments: | |
<genome_index_base> | 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. 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 (or BOWTIE2_INDEXES) environment variable. Please note that it is highly recommended that a FASTA file with the sequence(s) the genome being indexed be present in the same directory with the Bowtie index files and having the name <genome_index_base>.fa. If not present, TopHat will automatically rebuild this FASTA file from the Bowtie index files. |
<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 |
-N/--read-mismatches | Final read alignments having more than these many mismatches are discarded. The default is 2. |
--read-gap-length | Final read alignments having more than these many total length of gaps are discarded. The default is 2. |
--read-edit-dist | Final read alignments having more than these many edit distance are discarded. The default is 2. |
--read-realign-edit-dist | 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. |
--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. The default is 50bp. |
--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 choose the alignments based on their alignment scores if there are more than this number. The default is 20 for read mapping. Unless you use --report-secondary-alignments, TopHat will report the alignments with the best alignment score. If there are more alignments with the same score than this number, TopHat will randomly report only this many alignments. In case of using --report-secondary-alignments, TopHat will try to report alignments up to this option value, and TopHat may randomly output some of the alignments with the same score to meet this number. |
--report-secondary-alignments | By default TopHat reports best or primary alignments based on alignment scores (AS). Use this option if you want to output additional or secondary alignments (up to 20 alignments will be reported this way, this limit can be changed by using the -g/--max-multihits option above). |
--no-discordant | For paired reads, report only concordant mappings. |
--no-mixed | For paired reads, only report read alignments if both reads in a pair can be mapped (by default, if TopHat cannot find a concordant or discordant alignment for both reads in a pair, it will find and report alignments for each read separately; this option disables that behavior). |
--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 micro-exons. Works only for reads 50bp or longer. |
--library-type | The default is unstranded (fr-unstranded). If either fr-firststrand or fr-secondstrand is specified, every read alignment will have anXS attribute tag as explained below. Consider supplying library type options below to select the correct RNA-seq protocol. |
Library Type | Examples | Description |
fr-unstranded | Standard Illumina | Reads 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-firststrand | dUTP, NSR, NNSR | Same 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-secondstrand | Ligation, Standard SOLiD | Same 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. |
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.
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).
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.
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.
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 |
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