TopHat是基于Bowtie的将RNA-Seq数据mapping到参考基因组上，从而鉴定可变剪切（exon-exon splice junctions）。

安装

最简单的安装方法，注意版本

• 下载Bowtie、TopHat、Cufflinks的二进制发布包，解压到相同的目录
• 下载samtools，make，将生成的可执行samtools程序也cp到同一个目录
• 增加该目录到PATH

参数与使用

Usage: tophat [options]* <index_base> <reads1_1[,…,readsN_1]> [reads1_2,…readsN_2]

• -o  输出目录，默认值为 “./tophat_out”。
• –solexa-quals/solexa1.3-quals 质量编码，关于质量编码格式请参考《Fastq格式详解》
• -p 线程数，默认值为单线程1.，可以使用多线程
• -G/–GTFSupply TopHat with a set of gene model annotations and/or known transcripts, as a GTF 2.2 or GFF3 formatted file.指定已有转录本信息
• –no-novel-juncs 不查找新的可变剪切
• -r 比对时两成对引物间的距离中值。比如说，如果你的插入片段有300bp，而每个引物有50bp，那么r值就应该是200=(300+50*2)/2。没有默认值，如果是末端配对比对时这个值是必须的。
• –mate-std-dev 末端配对时中间插入片段的长度的标准差，默认值为20bp

paired-end数据应该如何做

paired end reads是好还是坏，好又好在哪里？如何从结果中体现，如何同一批paired-end reads 使用paired-end 参数与不适用差别在哪里？

READS文件

paired end的reads必须放在不同的两个文件中，文件名必须按照*_1, *_2的规范成对出现，Mixing paired- and single- end reads together is not supported.不要将paired-end的数据与single end reads放到一起进行处理。

设置-R参数

大多数情况，使用默认值就可以了，TopHat允许一定量的偏差，TopHat在多个地方使用到这个值，比如当寻找剪切位点与fusion break point。同时在生成报告的最后阶段选择最佳的alignment时，用到这个信息。可以先用少量的数据进行比对，在比对结果的SAM结果中，对于paired reads，第九列是插入片段的大概长度，可以用这个数值减去两倍的read的长度，就可以得到实际的-r参数需要设置的大小，如果值太大应该小心，只有比对上同一个外显子的情况具有意义。

控制结果的参数

• –no-discordant For paired reads, report only concordant mappings.对于成对的读取，只报告一致的映射。
• –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 和谐 or discordant 不和谐 alignment for both reads in a pair, it will find and report alignments for each read separately分别; this option disables that behavior).

结果读取

常见的错误及其解决思路

[ERRNO 2]NO SUCH FILE OR DIRECTORY

[Thu Oct 18 17:20:15 2012] Beginning TopHat run (v1.3.2)

-----------------------------------------------

[Thu Oct 18 17:20:15 2012] Preparing output location ./tt/

[Thu Oct 18 17:20:15 2012] Checking for Bowtie index files

[Thu Oct 18 17:20:15 2012] Checking for reference FASTA file

[Thu Oct 18 17:20:15 2012] Checking for Bowtie

        Bowtie version:                  0.12.8.0

[Thu Oct 18 17:20:15 2012] Checking for Samtools

        Samtools Version: 0.1.18

[Thu Oct 18 17:20:15 2012] Generating SAM header for ./hg19/genome

[Thu Oct 18 17:20:38 2012] Preparing reads

        format:          fastq

        quality scale:   solexa33 (reads generated with GA pipeline version < 1.3)

[Thu Oct 18 17:20:38 2012] Reading known junctions from GTF file

        Left  reads: min. length=35, count=53327887

[Thu Oct 18 18:05:11 2012] Mapping left_kept_reads against genome with Bowtie

[Thu Oct 18 23:04:52 2012] Processing bowtie hits

[Thu Oct 18 23:52:29 2012] Mapping left_kept_reads_seg1 against genome with Bowtie (1/4)

[Fri Oct 19 03:59:45 2012] Mapping left_kept_reads_seg2 against genome with Bowtie (2/4)

[Fri Oct 19 08:04:09 2012] Mapping left_kept_reads_seg3 against genome with Bowtie (3/4)

[Fri Oct 19 11:55:20 2012] Mapping left_kept_reads_seg4 against genome with Bowtie (4/4)

[Fri Oct 19 15:37:53 2012] Searching for junctions via segment mapping

[Fri Oct 19 16:24:47 2012] Retrieving sequences for splices

[Fri Oct 19 16:30:15 2012] Indexing splices

[Fri Oct 19 16:30:41 2012] Mapping left_kept_reads_seg1 against segment_juncs with Bowtie (1/4)

[Fri Oct 19 17:05:17 2012] Mapping left_kept_reads_seg2 against segment_juncs with Bowtie (2/4)

[Fri Oct 19 17:39:56 2012] Mapping left_kept_reads_seg3 against segment_juncs with Bowtie (3/4)

[Fri Oct 19 18:13:04 2012] Mapping left_kept_reads_seg4 against segment_juncs with Bowtie (4/4)

[Fri Oct 19 18:43:58 2012] Joining segment hits

[Fri Oct 19 19:37:28 2012] Reporting output tracks

        [FAILED]

Error: [Errno 2] No such file or directory

[samopen] SAM header is present: 25 sequences.