Shulou(Shulou.com)06/02 Report--

In this issue, Xiaobian will bring you about how to use tophat-fusion to identify fusion genes. The article is rich in content and analyzed and described from a professional perspective. After reading this article, I hope you can gain something.

Tophat-fusion is a tool for identifying fusion genes using RNA_seq data.

The software is integrated in tophat software, only need to install tophat can be used, the use method is relatively simple, the only thing to pay attention to is the directory structure.

Tophat-fusion requires a fixed directory structure. For example, if I analyze tophat-fusion under the result folder, I need to prepare the following files under this directory.

Generef.txt and Geneens.txt for species, which can be downloaded from UCSC

Create a new blast folder. Note that the folder name must be "blast". Under the blast folder, you need to download all files starting with nt, human_genomic, other_genomic from NCBI. The download link is as follows:

ftp://ftp.ncbi.nlm.nih.gov/blast/db/

The output directory of the result, each sample corresponds to an output directory, the prefix of the output directory is tophat_, underlined followed by the sample name, similar to tophat_MCF,MCF is the sample name

Of course, you also need the index file of bowtie1 corresponding to the species. Note that the index of bowtie 1 must be recommended for tophat detection of fusion genes.

After the above documents are ready, you can start analyzing them. The steps are as follows

1. alignment reference genome

The first step is actually to use tophat to align reads to the reference genome, but for reads fused with genes, the alignment method is special, and additional parameters need to be added. The specific code is as follows

tophat2 -o tophat_MCF7 -p 20 --fusion-search --keep-fasta-order --bowtie1 --no-coverage-search -r 0 --mate-std-dev 80 --max-intron-length 100000 --fusion-min-dist 100000 --fusion-anchor-length 13 --fusion-ignore-chromosomes chrM hg19_bowtie1/hg19 SRR064286_1.fastq SRR064286_2.fastq2. generate results

In the result directory, run the following code directly

tophat-fusion-post -p 20 --num-fusion-reads 1 --num-fusion-pairs 2 --num-fusion-both 5 hg19_bowtie1/hg19

By default, human fusion genes are processed. If it is other species, you need to add the--non-human parameter.

Tophat-fusion will automatically identify the corresponding samples according to the directory structure. After running, it will generate a folder named tophatfusion_out, which contains the fusion gene analysis results of all samples.

We only need to look at the result.html file, the content is as follows

Each column has the following meaning

Sample name in which a fusion is identified

Gene on the "left" side of the fusion

Chromosome ID on the left

Coordinates on the left

Gene on the "right" side

Chromosome ID on the right

Coordinates on the right

Number of spanning reads

Number of spanning mate pairs

Number of spanning mate pairs where one end spans a fusion

Compared to fusionmap, the software runs exceptionally long.

The above is how to use tophat-fusion to identify fusion genes shared by everyone. If there are similar doubts, please refer to the above analysis for understanding. If you want to know more about it, please pay attention to the industry information channel.

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