KEGG数据库，即京都基因和基因组百科全书（Kyoto Encyclopedia of Genes and Genomes），是系统分析基因功能、基因组信息的数据库。

KofamKOALA是一个方便的KEGG功能注释工具，由创建KEGG的京都大学化研所生物信息中心学者在2019年11月发表于Bioinformatics。

以隐马尔科夫模型(HMM)创建的KOfam来进行蛋白序列同源搜索，其准确性可与性能最佳的工具相媲美， 有网页和Linux两个版本，本文重点介绍Linux版的安装与使用。

网页版

网址 https://www.genome.jp/tools/kofamkoala/

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网页填入蛋白序列信息，设值E值和留下邮箱点击Compute，只需要等待邮箱回复

Linux版

Linux版本的KofamKOALA 需要下载 KOfam（数据库）和 KofamScan（软件），软件依赖Ruby，HMMER和GNU Parallel（事先没有安装可以看以下教程）

安装

我们以kofamscan安装在主目录$HOME(或者叫~)下为例介绍：

step1

下载和解压 KOfam 和 KofamScan

mkdir -p ~/kofamscan/dbcd ~/kofamscan/dbwget ftp://ftp.genome.jp/pub/db/kofam/ko_list.gz wget ftp://ftp.genome.jp/pub/db/kofam/profiles.tar.gz gunzip ko_list.gz tar xvzf profiles.tar.gz mkdir -p ~/kofamscan/bincd ~/kofamscan/binwget ftp://ftp.genome.jp/pub/tools/kofamscan/kofamscan-1.2.0.tar.gz # 注意kofamscan版本tar xvzf kofamscan-1.2.0.tar.gz

step2

下载 Ruby  HMMER  GNU Parallel

cd ~/kofamscan mkdir ruby hmmer parallel src cd src# Ruby版本应不小于2.4,这里演示的是2.7版;HMMER应大于3.1，这里是3.3;Parallel为最新版wget https://cache.ruby-lang.org/pub/ruby/2.7/ruby-2.7.0.tar.gzwget http://eddylab.org/software/hmmer/hmmer-3.3.tar.gzwget ftp://ftp.gnu.org/gnu/parallel/parallel-latest.tar.bz2

安装 Ruby

cd ~/kofamscan/src tar xvzf ruby-2.7.0.tar.gz cd ruby-2.7.0./configure --prefix=$HOME/kofamscan/ruby make make install

安装 HMMER

cd ~/kofamscan/src tar xvzf hmmer-3.3.tar.gz cd hmmer-3.3./configure --prefix=$HOME/kofamscan/hmmer make make install

安装 GNU Parallel

cd ~/kofamscan/src tar xvjf parallel-latest.tar.bz2 cd parallel-20190322  # 这里根据版本而定./configure --prefix=$HOME/kofamscan/parallel make make install

将Ruby路径加入环境变量（之后执行如果报错可能是ruby的问题，推荐ruby按照本文方法安装）

export PATH=$HOME/kofamscan/ruby/bin:$PATH

step3

复制config-template.yml文件并重命名为config.yml

cd ~/kofamscan/bin/ cp config-template.yml config.yml

cat config.yml文件,内容如下:

我们需要在config.yml添加键值，以便ruby识别读取.

可以vim编辑加入以下内容到config.yml，注意绝对路径.

profile: /path/to/home/kofamscan/db/profiles ko_list: /path/to/home/kofamscan/db/ko_list hmmsearch: /path/to/home/kofamscan/hmmer/bin/hmmsearch parallel: /path/to/home/kofamscan/parallel/bin/parallel

如：

若hummsearch和parallel可安装在其他地方改为相关路径

使用

现在可以使用了，我们准备蛋白序列fasta文件（注意必须是蛋白序列，不支持核酸序列）

./exec_annotation -o result.txt query.fasta

如我在~/kofamscan/test/文件夹下有samples.fasta文件，定义输出文件为res.txt

cd ~/kofamscan/bin/kofamscan-1.2.0 # 路径中有exec_annotation文件./exec_annotation -o ~/kofamscan/test/res.txt~/kofamscan/test/samples.fasta

运行完毕后的输出文件：

若报错可能是ruby的路径不在首选环境变量，可执行：

export PATH=$HOME/kofamscan/ruby/bin:$PATH

./exec_annotation -h查看全部参数：

## Options
- `-o FILE`- The result are output to `FILE`. It defaults to `stdout`.
- `-p`, `--profile=PROFILE`- Use `PROFILE` as a profile database. See [Profiles](#profiles)
- `-k`, `--ko-list=FILE`- Use `FILE` as a KO list.
- `--cpu=N`- Set the number of `hmmsearch` processes started simultaneously to `N`. It defaults to 1 unless it is set in `config.yml`.
- `-c FILE`- Use `FILE` as a config file instead of `config.yml` in the same directory as `exec_annotation`.
- `--tmp-dir=DIR`- Use `DIR` as a temporary directory where hmmsearch results are. It will be created if not exist. It defaults to `./tmp`.
- `-E`, `--e-value=VALUE`- Require E-value to be smaller than or equal to `VALUE`. If not, an asterisk will not be added in `detail` format or the hit will not be reported in other formats.
- `-T`, `--threshold-scale=VALUE`- The score thresholds are multiplied by `VALUE`. For example, with `-T2` option, the thresholds become twice as strict.
- `-f`, `--format=FORMAT`- Set the format of the output to `FORMAT`. Three formats below are available.- `detail`- Default format. Gene name, assigned K number, threshold of the KO, hmmsearch score and E-value, and the definition of KO are shown. In addition, an asterisk '*' is added to the head of the line if the score is higher than the threshold.- `mapper`- Format which can be used for [KEGG Mapper](https://www.genome.jp/kegg/mapper.html) input. It includes a gene name and an assigned K number separated by a tab. Here, an assigned K number represents a hit with score above the predefined threshold. Note that for some KOs, predefined score thresholds are not available when they are represented by a very few number of sequences in KEGG GENES.- `mapper-oneline`- Similar to `mapper`, but when more than one KO are assigned to a gene, all assigned KO are shown in one line separated by tabs.
- `--[no-]report-unannotated`- With `--report-unannotated` option, gene names are shown even when no KO is assigned (default when `--format=mapper(-oneline)`). With `--no-report-unannotated` such genes are not shown at all (default when `--format=detail`).
- `--create-alignment`- `hmmsearch`'s normal outputs per profile are stored in the temporary directory. In addition, domain information and alignments in the outputs will be rearranged per query.- Not compatible with `--reannotation`
- `-r`, `--reannotation`- Skip `hmmsearch` and assume that `hmmsearch` outputs are already in the temporary directory. This will help you to make an output in a different format or redo annotation changing thresholds.- Not compatible with `--create-alignment`
- `-h`, `--help`- Show brief help message.

参考 ：

https://www.genome.jp/tools/kofamkoala/

https://academic.oup.com/bioinformatics/advance-article/doi/10.1093/bioinformatics/btz859/5631907

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