Kraken2是一款快速宏基因组DNA序列进行物种注释的软件。我将其用于测序数据以及序列的物种的物种注释，检查测序数据以及序列是否有污染。
conda以及github网址：
https://anaconda.org/bioconda/kraken2
https://github.com/DerrickWood/kraken2
官方帮助文档：
https://github.com/DerrickWood/kraken2/blob/master/docs/MANUAL.markdown
两种安装方式，我选择conda安装，提示我会改变环境中的软件，有的会被移除或者降级，于是改为创建一个环境。
kraken2安装
#直接在系统中安装
conda install -c bioconda kraken2
#创建一个新环境在环境中安装
conda create -n kraken2 -c bioconda kraken2
source activate kraken2
kraken2建库
kraken2 主要有两个命令一个是kraken2-build，kraken2。
进行筛选则需要现建立库，那我们先看kraken2-build
Usage: kraken2-build [task option] [options]
Task options (exactly one must be selected):
--download-taxonomy Download NCBI taxonomic information
--download-library TYPE Download partial library
(TYPE = one of "archaea", "bacteria", "plasmid",
"viral", "human", "fungi", "plant", "protozoa",
"nr", "nt", "UniVec", "UniVec_Core")
--special TYPE Download and build a special database
(TYPE = one of "greengenes", "silva", "rdp")
--add-to-library FILE Add FILE to library
--build Create DB from library
(requires taxonomy d/l'ed and at least one file
in library)
--clean Remove unneeded files from a built database
--standard Download and build default database
--help Print this message
--version Print version information
Options:
--db NAME Kraken 2 DB name (mandatory except for
--help/--version)
--threads # Number of threads (def: 1)
--kmer-len NUM K-mer length in bp/aa (build task only;
def: 35 nt, 15 aa)
--minimizer-len NUM Minimizer length in bp/aa (build task only;
def: 31 nt, 12 aa)
--minimizer-spaces NUM Number of characters in minimizer that are
ignored in comparisons (build task only;
def: 7 nt, 0 aa)
--protein Build a protein database for translated search
--no-masking Used with --standard/--download-library/
--add-to-library to avoid masking low-complexity
sequences prior to building; masking requires
dustmasker or segmasker to be installed in PATH,
which some users might not have.
--max-db-size NUM Maximum number of bytes for Kraken 2 hash table;
if the estimator determines more would normally be
needed, the reference library will be downsampled
to fit. (Used with --build/--standard/--special)
--use-ftp Use FTP for downloading instead of RSYNC; used with
--download-library/--download-taxonomy/--standard.
--skip-maps Avoids downloading accession number to taxid maps,
used with --download-taxonomy.
--load-factor FRAC Proportion of the hash table to be populated
(build task only; def: 0.7, must be
between 0 and 1).
--fast-build Do not require database to be deterministically
built when using multiple threads. This is faster,
but does introduce variability in minimizer/LCA
pairs. Used with --build and --standard options.
首先构建一个标准数据库看看吧，执行如下命令。
mkdir -p standard_db
kraken2-build --standard --threads 12 --db standard_db
出现以下报错的时候不要慌，有人已经解决了。
https://github.com/DerrickWood/kraken2/issues/508
rsync_from_ncbi.pl: unexpected FTP path (new server?) for https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/762/265/GCF_000762265.1_ASM762
报错调整
使用conda安装的话，出错的rsync_from_ncbi.pl在$your_path/anaconda3/envs/kraken2/libexec/rsync_from_ncbi.pl
把rsync_from_ncbi.pl文件的
if (! ($full_path =~ s#^ftp://${qm_server}${qm_server_path}/##))
换成
if (! ($full_path =~ s#^https://${qm_server}${qm_server_path}/##))
————————————————
原文链接：https://blog.csdn.net/weixin_52602016/article/details/120689897
构建完毕的标准库内应该以下文件
hash.k2d
library
opts.k2d
seqid2taxid.map
taxo.k2d
taxonomy
unmapped.txt
hash.k2d: 包含最小值到分类单元的映射
opts.k2d: 包含有关用于构建数据库的选项的信息
taxo.k2d: 包含用于构建数据库的分类信息
使用这条命令kraken2-build --standard --threads 12 --db standard_db按照其帮助文档应该是下载并建立索引库，但有时候会缺少以上几个文件可能是没有建立索引，可以用如下命令建立索引。
kraken2-build --build --threads 24 --db standard_db
整个下载以及构建时间是比较长的，我们可以在任务完成以后使用kraken2-inspect -db standard_db | head -5命令来检查一下数据库是否完整，如果正常会在屏幕上打印测试检索报告。
kraken2的库
我们可以使用kraken2-build --download-library $library_name下载其提供的库，并建立后续的索引库。
archaea: RefSeq complete archaeal genomes/proteins #古菌
bacteria: RefSeq complete bacterial genomes/proteins #细菌
plasmid: RefSeq plasmid nucleotide/protein sequences #质粒
viral: RefSeq complete viral genomes/proteins #病毒
human: GRCh38 human genome/proteins #人
fungi: RefSeq complete fungal genomes/proteins #真菌
plant: RefSeq complete plant genomes/proteins #植物
protozoa: RefSeq complete protozoan genomes/proteins #原始动物
nr: NCBI non-redundant protein database #非冗余蛋白库
nt: NCBI non-redundant nucleotide database #非冗余蛋白库
UniVec: NCBI-supplied database of vector, adapter, linker,
and primer sequences that may be contaminating sequencing
projects and/or assemblies #常用污染序列，如载体、头、引物等
UniVec_Core: A subset of UniVec chosen to minimize false positive
hits to the vector database #常用污染序列核心库
标准模式下建的数据库只包括archaea、bacteria、human、plasmid、UniVec_Core、viral。
可以给一个db库下载多个数据
kraken2-build --download-library archaea --db $DBNAME
kraken2-build --download-library viral --db $DBNAME
添加序列数据，也可以根据需要添加自己的序列，但是需要根据Kraken2修改fasta序列的抬头信息>序列号|kraken:taxid|NCBI的分类好（详细查看官方帮助文档)
数据库序列准备完毕以后，就可建立索引了
kraken2-build --build --db $DBNAME
建立好索引以后就可以使用了。