基于conda环境使用mamba/conda安装配置QIIME 2 2023.9 Amplicon扩增子分析环境，q2cli主要功能模块介绍及使用

QIIME 2 2023.9 Amplicon Distribution介绍：

概述

qiime团队专门针对高通量扩增子序列分析退出的conda集成环境，包括了主要和常见的扩增子分析模块，用户可以单独使用各个模块，也可以使用各模块组成不同的分析流程。从2023.09版本开始特意将扩增子和宏基因组shotgun分成了两个环境，可能一个环境集成太大了。这样安装应该更轻盈。

QIIME 2 2023.9 Amplicon Distribution

看看文章吧，超强的团队：Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2 | Nature Biotechnology

2023.9Amplicon分发版qiime2主要功能模块介绍： 

The 2023.9 release of the QIIME 2 Amplicon Distribution includes the QIIME 2 framework, q2cli (a QIIME 2 command-line interface) and the following plugins:

• q2-alignment

• q2-composition

• q2-cutadapt

• q2-dada2

• q2-deblur

• q2-demux

• q2-diversity

• q2-diversity-lib

• q2-emperor

• q2-feature-classifier

• q2-feature-table

• q2-fragment-insertion

• q2-longitudinal

• q2-metadata

• q2-phylogeny

• q2-quality-control

• q2-quality-filter

• q2-sample-classifier

• q2-taxa

• q2-types

• q2-vsearch

还有更多的模块，都可以单独安装，一般建议成套安装就行：

QIIME 2 Library

 

QIIME2的安装：

还是继续用conda吧，这个用起来也挺方便，docker稍微增加了点门槛，看个人喜好

 官方安装介绍链接：

先看介绍选择分发板，根据自己喜好安装：Installing QIIME 2 — QIIME 2 2023.9.2 documentation

这里选择amplicon扩增子分析的环境：

查看安装指引：

Natively installing QIIME 2 — QIIME 2 2023.9.2 documentation

 选择amplicon分发版的linux环境配置指导：

下载环境依赖文件并安装： 

# 下载环境依赖配置文件
wget https://data.qiime2.org/distro/amplicon/qiime2-amplicon-2023.9-py38-linux-conda.yml# 使用mamba或conda 安装 ，-n后的名字自己定义就行
mamba env create -n qiime2-amplicon-2023.9 --file qiime2-amplicon-2023.9-py38-linux-conda.yml

要升级qiime2？，建议按日期名称命名各个版本重新安装，不需要的就直接删除旧的环境，因为有些旧的环境下的模块大家可能还需要用到，而在新的环境下可能已经废弃或更新了，所以建议重新配置安装新的conda环境就行。

开始使用qiime2 amplicon分发版 

要看怎么使用，或纠错，这里应该是最全的了，User Support - QIIME 2 Forum

这里来自刘永鑫团队的扩增子分析流程大家可以参考，把其中对应的步骤改为使用qiime模块分析即可：

使用vsearch进行16s扩增子高通量序列分析步骤-CSDN博客

EasyAmplicon (易扩增子)-扩增子高通量序列分析软件流程及脚本-详细使用方法——来自刘永鑫团队的秘籍-CSDN博客

当然qiime2 不仅仅提供分析模块，还开发了很多workflow，一个命令就可以按照workflow来得到结果了；可视化也有流程，后面大家看着参考：

 Overview of QIIME 2 Plugin Workflows — QIIME 2 2023.9.2 documentation

先激活环境并查看环境信息

# 激活环境，conda或mamba
mamba activate qiime2-amplicon-2023.9# 查看帮助信息
qiime --help

开始qiime2分析

分析前建议大家先建立独立的工作目录，并进入工作目录再开始操作：

1、导入数据：

查看帮助：

qiime tools import --helpUsage: qiime tools import [OPTIONS]Import data to create a new QIIME 2 Artifact. See https://docs.qiime2.org/for usage examples and details on the file types and associated semantictypes that can be imported.Options:--type TEXT             The semantic type of the artifact that will becreated upon importing. Use --show-importable-typesto see what importable semantic types are availablein the current deployment.                [required]--input-path PATH       Path to file or directory that should be imported.[required]--output-path ARTIFACT  Path where output artifact should be written.[required]--input-format TEXT     The format of the data to be imported. If notprovided, data must be in the format expected by thesemantic type provided via --type.--show-importable-types Show the semantic types that can be supplied to--type to import data into an artifact.--show-importable-formatsShow formats that can be supplied to --input-formatto import data into an artifact.--help                  Show this message and exit.

 大家可以先使用样例文件做为参考，然后按样例文件整理自己的数据

双端序列导入（混合样品测序，序列文件带barcode，分析时需要导入样品的metadata）Multiplexed paired-end FASTQ with barcodes in sequence

这种应该符合大家现在常规混合样品测序的情况了。

 metadata格式，重要的是第一列和第二列，或者再加一列barcode2 

 序列文件导入（三个文件，forward.fastq.gz, reverse.fastq.gz, sample_metadata.tsv）：

qiime tools import \--type MultiplexedPairedEndBarcodeInSequence \--input-path muxed-pe-barcode-in-seq \--output-path multiplexed-seqs.qza

单端序列导入（序列文件与barcode文件各自独立）

复制官方代码时注意删除上面的引号，这个复制过来容易变成中文的双引号，另外还需要注意存储路径的设置 ：

获取样例文件 

# 进入工作目录
cd emp-single-end-sequences/# barcode文件
wget \-O barcodes.fastq.gz \https://data.qiime2.org/2023.9/tutorials/moving-pictures/emp-single-end-sequences/barcodes.fastq.gz# 序列文件，这里是单端序列
wget \-O sequences.fastq.gz \https://data.qiime2.org/2023.9/tutorials/moving-pictures/emp-single-end-sequences/sequences.fastq.gz

查看要导入的文件格式：

# 
zcat barcodes.fastq.gz | head -n 10
# barcode文件内容，
@HWI-EAS440_0386:1:23:17547:1423#0/1
ATGCAGCTCAGT
+
IIIIIIIIIIIH
@HWI-EAS440_0386:1:23:14818:1533#0/1
CCCCTCAGCGGC
+
DDD@D?@B<<+/
@HWI-EAS440_0386:1:23:14401:1629#0/1
GACGAGTCAGTC#
zcat sequences.fastq.gz | head -n 10# sequences文件内容
@HWI-EAS440_0386:1:23:17547:1423#0/1
TACGNAGGATCCGAGCGTTATCCGGATTTATTGGGTTTAAAGGGAGCGTAGATGGATGTTTAAGTCAGTTGTGAAAGTTTGCGGCTCAACCGTAAAATTGCAGTTGATACTGGATATCTTGAGTGCAGTTGAGGCAGGGGGGGATTGGTGTG
+
IIIE)EEEEEEEEGFIIGIIIHIHHGIIIGIIHHHGIIHGHEGDGIFIGEHGIHHGHHGHHGGHEEGHEGGEHEBBHBBEEDCEDDD>B?BE@@B>@@@@@CB@ABA@@?@@=>?08;3=;==8:5;@6?######################
@HWI-EAS440_0386:1:23:14818:1533#0/1
CCCCNCAGCGGCAAAAATTAAAATTTTTACCGCTTCGGCGTTATAGCCTCACACTCAATCTTTTATCACGAAGTCATGATTGAATCGCGAGTGGTCGGCAGATTGCGATAAACGGGCACATTAAATTTAAACTGATGATTCCACTGCAACAA
+
64<2$24;1)/:*B<?BBDDBBD<>BDD############################################################################################################################
@HWI-EAS440_0386:1:23:14401:1629#0/1
TACGNAGGATCCGAGCGTTATCCGGATTTATTGGGTTTAAAGGGAGCGTAGGCGGACGCTTAAGTCAGTTGTGAAAGTTTGCGGCTCAACCGTAAAATTGCAGTTGATACTGGGTGTCTTGAGTACAGTAGAGGCAGGGGGGGGGTTGGGGG

## 激活 qiime2环境：## 导入数据
qiime tools import \--type EMPSingleEndSequences \--input-path ./ \--output-path emp-single-end-sequences.qza#### qiime tools import --show-importable-types  这个命令已经废弃了，大家注意在新版里没法用qiime tools list-types
Usage: qiime tools list-types [OPTIONS] [QUERIES]...List the available semantic types.Options:--strict    Show only exact matches for the type argument(s).--tsv       Print as machine readable tab-separated values.--help      Show this message and exit.

双端序列导入（序列文件与barcode文件各自独立）

获取样例文件  

cd emp-paired-end-sequences
# 下载正向序列
wget \-O forward.fastq.gz \https://data.qiime2.org/2023.9/tutorials/atacama-soils/1p/forward.fastq.gz#下载反向序列
wget \-O reverse.fastq.gz \https://data.qiime2.org/2023.9/tutorials/atacama-soils/1p/reverse.fastq.gz# 下载barcodes文件
wget \-O barcodes.fastq.gz \https://data.qiime2.org/2023.9/tutorials/atacama-soils/1p/barcodes.fastq.gz

# 查看输入文件格式
# 正向序列文件内容
zcat forward.fastq.gz | head -n 10
@M00176:65:000000000-A41FR:1:1101:14282:1412 1:N:0:0
NACGTAGGGTGCAAGCGTTAATCGGAATTACNGGNNNTAAAGCGTGCNNAGGCNNNNNNNNNNNANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
+
#>>>AAF@ACAA4BGCEEECGGHGGEFCFBG#BA###BABAEFGEEE##BBAA###########B######################################################################################
@M00176:65:000000000-A41FR:1:1101:16939:1420 1:N:0:0
NACGTAGGGGGCAAGCGTTGTCCGGAATCATTGGNNGTAAAGAGCGTGNAGGCNNNNNGNNANNTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
+
#>>ABAF@ABBBGGGGEEFGGGHGGFFGFHHHHH##BBEFDHHCGGFG#ABFF#####B##B##B######################################################################################
@M00176:65:000000000-A41FR:1:1101:14746:1560 1:N:0:0
TACGTAGGGAGCTAGCGTTGTCCGGAATCATTGGGCGTAAAGCGCGCGTAGGCGGCCAGATAAGTCCGGTGTAAAAGCCACAGGCTNNNNNNNNNNNNNNNNCNGGANNNNNNNNNNNNNNNNNNNNNNANNNNNNNNNNNNANNNNNGGN# 反向序列文件内容
zcat reverse.fastq.gz | head -n 10
@M00176:65:000000000-A41FR:1:1101:14282:1412 2:N:0:0
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCGGAATNCCATNCCNCTCTGCNNNNNNNNNNNNNNNNNNNNNNNNN
+
#########################################################################################################<</?F/#/</?#?<#<???/<#########################
@M00176:65:000000000-A41FR:1:1101:16939:1420 2:N:0:0
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCNCNNTNCNNNNNNNNNNNGGGAATTCCACNCGCCTCTTCNNNNNNNNNNNNNNNNNNNNNNNNN
+
######################################################################################?#?##?#?###########<??/?FC>FBF#</??FAGHG#########################
@M00176:65:000000000-A41FR:1:1101:14746:1560 2:N:0:0
NNNNTTTCGTNGATNNNNNNNCNGNNNNNNNNNNNNNNNNNNNTNNNNNNNTACCNNNNNNNNTGGGCCTCTGTGCGCATACCGTTCGAGAGCGCCGGCCACGGCATTCGTGTTGCTCCTCTAAGCTGTGCGTGTTCACTGTACAACCNNN# barcode文件内容
zcat barcodes.fastq.gz | head -n 10
@M00176:65:000000000-A41FR:1:1101:14282:1412 1:N:0:0
NNNNNNNNNNNN
+
############
@M00176:65:000000000-A41FR:1:1101:16939:1420 1:N:0:0
NNNNNNNNNNNN
+
############
@M00176:65:000000000-A41FR:1:1101:14746:1560 1:N:0:0
NNNNNNNNNNNN

数据导入：

cd emp-paired-end-sequencesqiime tools import \--type EMPPairedEndSequences \--input-path ./ \--output-path emp-paired-end-sequences.qza

带barcodes的单端序列文件导入

获取样例文件 

#
cd muxed-se-barcode-in-seq/
wget \-O sequences.fastq.gz \https://data.qiime2.org/2023.9/tutorials/importing/muxed-se-barcode-in-seq.fastq.gz

# 查看输入文件格式：
zcat sequences.fastq.gz | head -n 20
@M00899:113:000000000-A5K20:1:1101:18850:2539 1:N:0:2
GCTACGGGGGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCAACGCCGCGTGAACGATGAAGGTCTTCGGATCGTAAAGTTCTGTTGCAGGGGAAGATAATGACGGTACCCTGTGAGGAAGCCCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGCTAGCGTTATCCGGATTTACTGGGCGTAAAGGGTGCGTAGGTGGTCCTTCAAGTCGGTGGTTAAAGGCTAAGGCTCAACCGTAGTAAGCCGCCGAAACTGGAGGACTTGAGTGAAGGAGAGG
+
-8ABCC>=>5811884:<:99=?@EECFFGDFADECFFFEEDDEFEDEDFFFEEFCCBCF>CCB3CFF:BBFFFCCD,8@9C@C:+5@@:A@C<FDCFBEG>FFFDGCCEC?FGGGGGGGGGCFGGFCFGGGGGGGGGGEG7CFFGFFFGGGFG?FACE;:8CCCCEEF9<F@FFEGGC**/:3:2CC@:C;C81;C9<?>FF8C758CGGG2:7DC>EECEFE9+27CF492/8B7>D)7@F=FFCFF*9F52<2,289<0:44AB<49(3<>F51).69D?D34*44:4<5<B?::086
@M00899:113:000000000-A5K20:1:1101:25454:3578 1:N:0:2
CCTACGGGAGGCAGCAGTGAGGAATATTGGTCAATGGGCGAGAGCCTGAACCAGCCAAGTAGCGTGCAGGATGACGGCCCTATGGGTTGTAAACTGCTTTTGTATGGGGATAAAGTCAGTCACGTGTGATTGTTTGCAGGTACCATACGAATAAGGACCGGCTAATTCCGTGCCAGCAGCCGCGGTAATACGGAAGGTCCGGGCGTTATCCGGATTTATTGGGTTTAAAGGGAGCGTAGGCTGGAGATTAAGTGTGTTGTGAAATGTAGACGCTCAACGTCTGACTTGCAGCGCATACTGG
+
8ACCCGD@AA=18=======;CEFGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGFGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGEGGGGGGFFF?FGGGGGGGGEGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGCGFGGGFFFGGGGGGEFGGGGGGGGGGGGCGEGGDGGGGGGGGGG=CGGCECDGGFGGGGGGGGFGGGF>C>BFFEGFFFFF:FGGF=6=6:AFBGFFFFFFA9A<AFB?@0)>C:0<CF?C46FAD<??90;::?DA>
@M00899:113:000000000-A5K20:1:1101:25177:3605 1:N:0:2
CCTACGGGAGGCAGCAGTGAGGAATATTGGTCAATGGACGGAAGTCTGAACCAGCCAAGTAGCGTGCAGGATGACGGCCCTATGGGTTGTAAACTGCTTTTGTATGGGGATAAAGTTAGGGACGTGTCCCTATTTGCAGGTACCATACGAATAAGGACCGGCTAATTCCGTGCCAGCAGCCGCGGTAATACGGAAGGTCCAGGCGTTATCCGGATTTATTGGGTTTAAAGGGAGCGTAGGCTGGAGATTAAGTGTGTTGTGAAATGTAGACGCTCAACGTCTGAATTGCAGCGCATACTGG
+
88BCCEDAD9018======;;CCFGGGGFGGGFGGGGGGGGGGGGGGGGGGGGGGGFGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGFGGGGGGGGGGGGGFGGGGGGGGGGGGGGGGEGDGGGGGGGGGFFGGGGGGGGGFGGGFGGGFGGGFFGCGGGGGGFGGFDGGGGGGGGGGGGG5CBFGCGGGGC?FGGGGGGGGGGGDEGDDDGFGGGGGEGGGGGGA39>BFFDDEF4:D5@CE?CFFF>>ABGFFF9<A246<<<<B::DE=<?FGGFG4>F?DF?02211:DAF7### barcode已经在序列中

导入文件:

qiime tools import \--type MultiplexedSingleEndBarcodeInSequence \--input-path sequences.fastq.gz \--output-path multiplexed-seqs.qza

当然还有其他可导入文件格式，慢慢看吧，个人觉得有需要的时候查一下就行，不用全部了解：

qiime tools list-formats --importable

2、导出数据

官方示例，大家可以下载下来测试一下就行了，不再细讲了。

 下载测试：

wget \-O "feature-table.qza" \"https://data.qiime2.org/2023.9/tutorials/exporting/feature-table.qza"wget \-O "unrooted-tree.qza" \"https://data.qiime2.org/2023.9/tutorials/exporting/unrooted-tree.qza"

qiime tools export \--input-path feature-table.qza \--output-path exported-feature-table## 导出为biom格式文件
## 参考：https://biom-format.org/documentation/format_versions/biom-2.1.htmlqiime tools export \--input-path unrooted-tree.qza \--output-path exported-tree## 导出为.nwk文件，可在各类软件再直接出发育树的图
head tree.nwk 
(((New.CleanUp.ReferenceOTU1480:0.11995,(New.CleanUp.ReferenceOTU202:0.04479,New.CleanUp.ReferenceOTU432:0.0049)0.769:0.04661)1:0.26705,((New.CleanUp.ReferenceOTU1150:0.00016,(New.CleanUp.ReferenceOTU782:0.04264,(New.CleanUp.ReferenceOTU643:0.10438,(((New.CleanUp.ReferenceOTU1014:0.01521,New.CleanUp.ReferenceOTU270:0.02738)0.879:0.02315,(((New.CleanUp.ReferenceOTU1008:0.0378,(New.CleanUp.ReferenceOTU1222:0.01621,(New.CleanUp.ReferenceOTU230:0.01829,(New.CleanUp.ReferenceOTU1047:0.0303,New.CleanUp.ReferenceOTU605:0.00015)0.88:0.01596)0.871:0.01515)0.714:0.01214)0.996:0.06757,(New.CleanUp.ReferenceOTU681:0.00441,(New.CleanUp.ReferenceOTU1485:0.01177,New.CleanUp.ReferenceOTU130:0.05699)0.923:0.02883)0.367:0.02397)0.17:0.00665,(New.CleanUp.ReferenceOTU1330:0.0258,(New.CleanUp.ReferenceOTU903:0.04234,(((New.CleanUp.ReferenceOTU1077:0.00014,(New.CleanUp.ReferenceOTU582:0.0171,New.CleanUp.ReferenceOTU987:0.0226)0.951:0.02372)0.892:0.02206,(New.CleanUp.ReferenceOTU891:0.01038,(((New.CleanUp.ReferenceOTU1066:0.01299,New.CleanUp.ReferenceOTU764:0.02254)0.851:0.01818,(New.CleanUp.ReferenceOTU180:0.02021,New.CleanUp.ReferenceOTU535:0.00014)0.795:0.01084)0.921:0.02441,(New.CleanUp.ReferenceOTU1212:0.00521mkdir extracted-feature-table
qiime tools extract \--input-path feature-table.qza \--output-path extracted-feature-table## 

3、样品元数据处理

这个可能得科学上网，给大家下载了，大家参考下载吧。

https://download.csdn.net/download/zrc_xiaoguo/88616825?spm=1001.2014.3001.5503

样例文件列表：

sample-metadata数据：

元数据表qiime2转换命令：

qiime metadata tabulate \--m-input-file sample-metadata.tsv \--o-visualization tabulated-sample-metadata.qzvqiime metadata tabulate \--m-input-file faith_pd_vector.qza \--o-visualization tabulated-faith-pd-metadata.qzvqiime metadata tabulate \--m-input-file sample-metadata.tsv \--m-input-file faith_pd_vector.qza \--o-visualization tabulated-combined-metadata.qzvqiime emperor plot \--i-pcoa unweighted_unifrac_pcoa_results.qza \--m-metadata-file sample-metadata.tsv \--m-metadata-file faith_pd_vector.qza \--o-visualization unweighted-unifrac-emperor-with-alpha.qzv

转换后数据qzv查看

4、数据过滤

Filtering data — QIIME 2 2023.9.2 documentation

特征表过滤Filtering feature tables

基于总出现频率过滤，去掉低丰度序列，或筛选一定丰度的序列，比如说将频率低于1500的序列去掉，包括两个参数，最高和最低频率

The --p-min-frequency and --p-max-frequency can be combined to filter based on lower and upper limits of total frequency

这里只给了一个最低频率的限制。

qiime feature-table filter-samples \--i-table table.qza \--p-min-frequency 1500 \--o-filtered-table sample-frequency-filtered-table.qza

基于出现的偶然性的过滤？？？Contingency-based filtering，也就是最少在多少个样品中都需要出现，通量有最高和最低频率设定： --p-min-features and --p-min-samples。

qiime feature-table filter-features \--i-table table.qza \--p-min-samples 2 \--o-filtered-table sample-contingency-filtered-table.qza

 字段筛选Identifier-based filtering， 也就是保留哪些特征数据内容。

qiime feature-table filter-samples \--i-table table.qza \--m-metadata-file samples-to-keep.tsv \--o-filtered-table id-filtered-table.qza

同样还有很多其他的筛选方式：

Metadata-based filtering

Taxonomy-based filtering of tables and sequences

序列过滤Filtering sequences

比如说这里根据原序列文件和分类文件提取mitochondria,chloroplast这两个门水平的序列文件。

qiime taxa filter-seqs \--i-sequences sequences.qza \--i-taxonomy taxonomy.qza \--p-include p__ \--p-exclude mitochondria,chloroplast \--o-filtered-sequences sequences-with-phyla-no-mitochondria-no-chloroplast.qza

Filtering distance matrices

qiime diversity filter-distance-matrix \--i-distance-matrix distance-matrix.qza \--m-metadata-file samples-to-keep.tsv \--o-filtered-distance-matrix identifier-filtered-distance-matrix.qzaqiime diversity filter-distance-matrix \--i-distance-matrix distance-matrix.qza \--m-metadata-file sample-metadata.tsv \--p-where "[subject]='subject-2'" \--o-filtered-distance-matrix subject-2-filtered-distance-matrix.qza

5、重要参考数据库

这个不多说了吧，大家按使用需求来下载，后面逐步会用到：

Data resources — QIIME 2 2023.9.2 documentation

 6、各个插件模块使用

不熟悉的先查看help信息和官网介绍：

• q2-alignment

q2-alignment 是 QIIME 2 生态系统中的一个插件，用于对生物信息学序列数据进行比对和序列比较的工具。它可以用于将不同样本中的序列进行比对，分析它们之间的相似性和差异性。q2-alignment 提供了一些常用的比对算法和工具，使用户能够对序列进行比对、生成比对结果和后续的分析。

以下是 q2-alignment 插件的一些主要功能和使用方法：

功能和用途：

1. 序列比对：对DNA、RNA或蛋白质序列进行比对。
2. 多序列比对：能够处理多个序列文件，比对它们之间的相似性。
3. 生成比对结果：产生比对结果，以便后续分析或可视化。
4. 支持多种比对算法：包括常用的比对算法，如BLAST、MUSCLE、MAFFT 等。

qiime alignment --help
Usage: qiime alignment [OPTIONS] COMMAND [ARGS]...Description: This QIIME 2 plugin provides support for generating andmanipulating sequence alignments.Plugin website: https://github.com/qiime2/q2-alignmentGetting user support: Please post to the QIIME 2 forum for help with thisplugin: https://forum.qiime2.orgOptions:--version            Show the version and exit.--example-data PATH  Write example data and exit.--citations          Show citations and exit.--help               Show this message and exit.Commands:mafft      De novo multiple sequence alignment with MAFFTmafft-add  Add sequences to multiple sequence alignment with MAFFT.mask       Positional conservation and gap filtering.

# 运行比对（示例使用 MAFFT）
qiime alignment mafft \--i-sequences sequences.qza \--o-alignment aligned_sequences.qza

• q2-composition

用于进行组成分析，尤其是在处理微生物组数据中的相对丰度数据时非常有用。该插件可以帮助用户探索和比较微生物组中微生物群落的组成。

qiime composition --help
Usage: qiime composition [OPTIONS] COMMAND [ARGS]...Description: This QIIME 2 plugin supports methods for compositional dataanalysis.Plugin website: https://github.com/qiime2/q2-compositionGetting user support: Please post to the QIIME 2 forum for help with thisplugin: https://forum.qiime2.orgOptions:--version            Show the version and exit.--example-data PATH  Write example data and exit.--citations          Show citations and exit.--help               Show this message and exit.Commands:add-pseudocount  Add pseudocount to table.ancom            Apply ANCOM to identify features that differ in abundance.ancombc          Analysis of Composition of Microbiomes with Bias Correctionda-barplot       Differential abundance bar plotstabulate         View tabular output from ANCOM-BC.

# 导入数据
qiime composition add-pseudocount \--i-table table.qza \--o-composition-table composition.qza#数据转换
qiime composition ilr-transform \--i-table composition.qza \--o-transformed-table ilr_composition.qza#可视化分析结果
qiime composition pcoa \--i-table ilr_composition.qza \--o-pcoa ilr_composition_pcoa.qzaqiime emperor plot \--i-pcoa ilr_composition_pcoa.qza \--m-metadata-file metadata.txt \--o-visualization ilr_composition_emperor.qzv

 q2-cutadapt

q2-cutadapt插件是用于对DNA序列数据进行预处理和过滤的工具。它基于Cutadapt软件，允许用户对Illumina测序数据进行裁剪（trimming）、过滤（filtering）和修剪（adapter removal）等操作，以消除低质量序列、去除适配器、修剪序列末端等。这有助于提高序列数据的质量，为后续的分析准备干净、高质量的数据。

#
qiime cutadapt --help
Usage: qiime cutadapt [OPTIONS] COMMAND [ARGS]...Description: This QIIME 2 plugin uses cutadapt to work with adapters (e.g.barcodes, primers) in sequence data.Plugin website: https://github.com/qiime2/q2-cutadaptGetting user support: Please post to the QIIME 2 forum for help with thisplugin: https://forum.qiime2.orgOptions:--version            Show the version and exit.--example-data PATH  Write example data and exit.--citations          Show citations and exit.--help               Show this message and exit.Commands:demux-paired  Demultiplex paired-end sequence data with barcodes in-sequence.demux-single  Demultiplex single-end sequence data with barcodes in-sequence.trim-paired   Find and remove adapters in demultiplexed paired-endsequences.trim-single   Find and remove adapters in demultiplexed single-endsequences.

qiime cutadapt trim-paired \--i-demultiplexed-sequences demux.qza \--p-cores 8 \--p-front-f CCTACGGGNGGCWGCAG \--p-front-r GACTACHVGGGTATCTAATCC \--p-discard-untrimmed \--o-trimmed-sequences demux_trimmed.qza

参数解释：
--i-demultiplexed-sequences: 输入的序列文件（需提前导入到QIIME 2）。
--p-cores: 并行处理的CPU核心数量。
--p-front-f 和 --p-front-r: 正向和反向引物序列。
--p-discard-untrimmed: 丢弃未被修剪的序列。
--o-trimmed-sequences: 输出修剪后的序列文件。 

• q2-dada2

q2-dada2是QIIME 2的插件之一，基于DADA2算法，用于去噪和分析Illumina测序生成的16S rRNA数据。

qiime dada2 --help
Usage: qiime dada2 [OPTIONS] COMMAND [ARGS]...Description: This QIIME 2 plugin wraps DADA2 and supports sequence qualitycontrol for single-end and paired-end reads using the DADA2 R library.Plugin website: http://benjjneb.github.io/dada2/Getting user support: Please post to the QIIME 2 forum for help with thisplugin: https://forum.qiime2.orgOptions:--version            Show the version and exit.--example-data PATH  Write example data and exit.--citations          Show citations and exit.--help               Show this message and exit.Commands:denoise-ccs     Denoise and dereplicate single-end Pacbio CCSdenoise-paired  Denoise and dereplicate paired-end sequencesdenoise-pyro    Denoise and dereplicate single-end pyrosequencesdenoise-single  Denoise and dereplicate single-end sequences

qiime dada2 denoise-single \--i-demultiplexed-seqs input-demux.qza \--p-trim-left 0 \--p-trunc-len 120 \--o-representative-sequences rep-seqs-dada2.qza \--o-table table-dada2.qza \--o-denoising-stats stats-dada2.qza

 参数说明：

• --i-demultiplexed-seqs：输入的demultiplexed序列文件。
• --p-trim-left：要去除的序列的前部分碱基数量。
• --p-trunc-len：截断序列的长度。
• --o-representative-sequences：输出的代表序列文件。
• --o-table：生成的特征表文件。
• --o-denoising-stats：生成的去噪统计文件。

• q2-deblur

q2-deblur插件通过识别和去除16S rRNA基因测序数据中的测序错误和噪声，以生成高质量的序列数据。其主要步骤包括：

1. 生成特征表（Feature table）： 从原始的FASTQ格式文件中导入数据，创建特征表。
2. 质量过滤： 过滤低质量序列，去除低质量序列读数。
3. 去噪处理： 使用Deblur算法去除测序错误和噪声，生成高质量的特征序列。
4. 生成结果： 输出一个经过去噪处理和质量过滤的特征表和序列文件。

qiime deblur --help
Usage: qiime deblur [OPTIONS] COMMAND [ARGS]...Description: This QIIME 2 plugin wraps the Deblur software for performingsequence quality control.Plugin website: https://github.com/biocore/deblurGetting user support: Please post to the QIIME 2 forum for help with thisplugin: https://forum.qiime2.orgOptions:--version            Show the version and exit.--example-data PATH  Write example data and exit.--citations          Show citations and exit.--help               Show this message and exit.Commands:denoise-16S      Deblur sequences using a 16S positive filter.denoise-other    Deblur sequences using a user-specified positive filter.visualize-stats  Visualize Deblur stats per sample.

qiime deblur denoise-16S \--i-demultiplexed-seqs your_qza_file.qza \--p-trim-length 250 \  # 设置序列截断长度--o-representative-sequences rep_seqs.qza \--o-table table.qza \--p-sample-stats \     # 如果需要生成样本统计信息--o-stats deblur_stats.qza

 结果数据导出

# 结果数据导出
qiime tools export --input-path rep_seqs.qza --output-path exported_rep_seqs
qiime tools export --input-path table.qza --output-path exported_table
qiime tools export --input-path deblur_stats.qza --output-path exported_stats

• q2-demux

q2-demux 插件用于处理 DNA 或 RNA 测序数据的样本数据解复用（demultiplexing）和质量控制。这个插件允许用户根据样本的不同 DNA 或 RNA 序列标签（barcode 或者 Illumina 测序的 index）将混合测序数据集拆分成单独的样本。下面是 q2-demux 插件的基本介绍和使用步骤：

q2-demux 插件功能：

1. 数据解复用（Demultiplexing）：

   • 将混合测序数据根据每个样本的唯一标识（barcode 或 index）分割成单独的样本序列文件。

2. 质量控制：

   • 提供了检查序列数据质量的功能，允许用户查看样本数据的质量分数并进行必要的处理，比如修剪或过滤低质量序列。

qiime demux --help
Usage: qiime demux [OPTIONS] COMMAND [ARGS]...Description: This QIIME 2 plugin supports demultiplexing of single-end andpaired-end sequence reads and visualization of sequence quality information.Plugin website: https://github.com/qiime2/q2-demuxGetting user support: Please post to the QIIME 2 forum for help with thisplugin: https://forum.qiime2.orgOptions:--version            Show the version and exit.--example-data PATH  Write example data and exit.--citations          Show citations and exit.--help               Show this message and exit.Commands:emp-paired                Demultiplex paired-end sequence data generatedwith the EMP protocol.emp-single                Demultiplex sequence data generated with the EMPprotocol.filter-samples            Filter samples out of demultiplexed data.partition-samples-paired  Split demultiplexed sequence data into partitions.partition-samples-single  Split demultiplexed sequence data into partitions.subsample-paired          Subsample paired-end sequences withoutreplacement.subsample-single          Subsample single-end sequences withoutreplacement.summarize                 Summarize counts per sample.tabulate-read-counts      Tabulate counts per sample

# 执行去重复
qiime demux emp-paired \--i-seqs demux.qza \--m-barcodes-file sample-metadata.tsv \--m-barcodes-column BarcodeSequence \--o-per-sample-sequences demux-paired-end.qza \--o-error-correction-details demux-details.qza## 查看结果
qiime demux summarize \--i-data demux-paired-end.qza \--o-visualization demux-summary.qzv

• q2-diversity

q2-diversity是其中一个用于计算和分析生物多样性的插件。它可以帮助你评估样本群落的多样性和差异性。

qiime diversity --help
Usage: qiime diversity [OPTIONS] COMMAND [ARGS]...Description: This QIIME 2 plugin supports metrics for calculating andexploring community alpha and beta diversity through statistics andvisualizations in the context of sample metadata.Plugin website: https://github.com/qiime2/q2-diversityGetting user support: Please post to the QIIME 2 forum for help with thisplugin: https://forum.qiime2.orgOptions:--version            Show the version and exit.--example-data PATH  Write example data and exit.--citations          Show citations and exit.--help               Show this message and exit.Commands:adonis                     adonis PERMANOVA test for beta group significancealpha                      Alpha diversityalpha-correlation          Alpha diversity correlationalpha-group-significance   Alpha diversity comparisonsalpha-phylogenetic         Alpha diversity (phylogenetic)alpha-rarefaction          Alpha rarefaction curvesbeta                       Beta diversitybeta-correlation           Beta diversity correlationbeta-group-significance    Beta diversity group significancebeta-phylogenetic          Beta diversity (phylogenetic)beta-rarefaction           Beta diversity rarefactionbioenv                     bioenvcore-metrics               Core diversity metrics (non-phylogenetic)core-metrics-phylogenetic  Core diversity metrics (phylogenetic and non-phylogenetic)filter-distance-matrix     Filter samples from a distance matrix.mantel                     Apply the Mantel test to two distance matricespartial-procrustes         Partial Procrustespcoa                       Principal Coordinate Analysispcoa-biplot                Principal Coordinate Analysis Biplotprocrustes-analysis        Procrustes Analysistsne                       t-distributed stochastic neighbor embeddingumap                       Uniform Manifold Approximation and Projection

# Alpha多样性（样本内部多样性）
# 计算Shannon指数：
qiime diversity alpha \--i-table your_feature_table.qza \--p-metric shannon \--o-alpha-diversity shannon_vector.qza#Beta多样性（样本间多样性）
# 计算Bray-Curtis距离：qiime diversity beta \--i-table your_feature_table.qza \--p-metric braycurtis \--o-distance-matrix braycurtis_distance_matrix.qza# 可视化和统计分析
qiime diversity alpha-group-significance \--i-alpha-diversity shannon_vector.qza \--m-metadata-file your_sample_metadata.txt \--o-visualization shannon_group_significance.qzv
qiime diversity beta-group-significance \--i-distance-matrix braycurtis_distance_matrix.qza \--m-metadata-file your_sample_metadata.txt \--o-visualization braycurtis_group_significance.qzv

• q2-diversity-lib

q2-diversity-lib 简介

q2-diversity-lib是QIIME 2的一个插件，用于计算多样性指数和样本间的差异。它基于多种生物多样性指标来评估微生物群落的多样性，并允许用户进行统计比较和可视化。

功能特性

1. 计算多样性指数：支持计算多种多样性指数，如Shannon、Simpson、Chao1等，可以帮助衡量群落内物种的多样性和丰富度。
2. Beta多样性计算：计算不同样本之间的差异和相似性，例如Bray-Curtis、Jaccard、Unweighted UniFrac、Weighted UniFrac等距离指标。
3. Beta多样性可视化：生成多样性分析的可视化图表，如PCoA（Principal Coordinates Analysis）图表，展示样本之间的差异。
4. 组间比较：支持组间多样性比较，通过PERMANOVA（Permutational Multivariate Analysis of Variance）等方法进行样本群落的差异性分析。

qiime diversity-lib --help
Usage: qiime diversity-lib [OPTIONS] COMMAND [ARGS]...Description: This QIIME 2 plugin computes individual metrics for  communityalpha and beta diversity.Plugin website: https://github.com/qiime2/q2-diversity-libGetting user support: Please post to the QIIME 2 forum for help with thisplugin: https://forum.qiime2.orgOptions:--version            Show the version and exit.--example-data PATH  Write example data and exit.--citations          Show citations and exit.--help               Show this message and exit.Commands:alpha-passthrough               Alpha Passthrough (non-phylogenetic)beta-passthrough                Beta Passthrough (non-phylogenetic)beta-phylogenetic-meta-passthroughBeta Phylogenetic Meta Passthroughbeta-phylogenetic-passthrough   Beta Phylogenetic Passthroughbray-curtis                     Bray-Curtis Dissimilarityfaith-pd                        Faith's Phylogenetic Diversityjaccard                         Jaccard Distanceobserved-features               Observed Featurespielou-evenness                 Pielou's Evennessshannon-entropy                 Shannon's Entropyunweighted-unifrac              Unweighted Unifracweighted-unifrac                Weighted Unifrac

计算使用 

#计算Alpha多样性（多样性指数）
qiime diversity alpha \--i-table table.qza \--p-metric shannon \--o-alpha-diversity shannon_alpha.qza#计算Beta多样性距离
qiime diversity beta \--i-table table.qza \--p-metric braycurtis \--o-distance-matrix braycurtis_distance.qza#可视化Beta多样性分析结果（例如PCoA）
qiime diversity pcoa \--i-distance-matrix braycurtis_distance.qza \--o-pcoa braycurtis_pcoa.qza
qiime emperor plot \--i-pcoa braycurtis_pcoa.qza \--m-metadata-file sample-metadata.tsv \--o-visualization braycurtis_emperor.qzv#进行组间多样性比较（以PERMANOVA为例）
qiime diversity beta-group-significance \--i-distance-matrix braycurtis_distance.qza \--m-metadata-file sample-metadata.tsv \--o-visualization braycurtis_permanova.qzv \--p-method permanova

• q2-emperor

qiime emperor --help
Usage: qiime emperor [OPTIONS] COMMAND [ARGS]...Description: This QIIME 2 plugin wraps Emperor and supports interactivevisualization of ordination plots.Plugin website: http://emperor.microbio.meGetting user support: Please post to the QIIME 2 forum for help with thisplugin: https://forum.qiime2.orgOptions:--version            Show the version and exit.--example-data PATH  Write example data and exit.--citations          Show citations and exit.--help               Show this message and exit.Commands:biplot           Visualize and Interact with Principal Coordinates AnalysisBiplotplot             Visualize and Interact with Principal Coordinates AnalysisPlotsprocrustes-plot  Visualize and Interact with a procrustes plot

• q2-feature-classifier

q2-feature-classifier 是 QIIME 2 中的一个插件，用于对16S rRNA或ITS等序列数据进行分类和注释。它主要用于将序列分类为特定的分类单元，比如对OTUs（操作分类单元）或者物种进行分类。

qiime feature-classifier --help
Usage: qiime feature-classifier [OPTIONS] COMMAND [ARGS]...Description: This QIIME 2 plugin supports taxonomic classification offeatures using a variety of methods, including Naive Bayes, vsearch, andBLAST+.Plugin website: https://github.com/qiime2/q2-feature-classifierGetting user support: Please post to the QIIME 2 forum for help with thisplugin: https://forum.qiime2.orgOptions:--version            Show the version and exit.--example-data PATH  Write example data and exit.--citations          Show citations and exit.--help               Show this message and exit.Commands:blast                           BLAST+ local alignment search.classify-consensus-blast        BLAST+ consensus taxonomy classifierclassify-consensus-vsearch      VSEARCH-based consensus taxonomy classifierclassify-hybrid-vsearch-sklearnALPHA Hybrid classifier: VSEARCH exact match+ sklearn classifierclassify-sklearn                Pre-fitted sklearn-based taxonomy classifierextract-reads                   Extract reads from reference sequences.find-consensus-annotation       Find consensus among multiple annotations.fit-classifier-naive-bayes      Train the naive_bayes classifierfit-classifier-sklearn          Train an almost arbitrary scikit-learnclassifiermakeblastdb                     Make BLAST database.vsearch-global                  VSEARCH global alignment search

#训练一个 SILVA 数据库上的分类器可以使用如下命令
qiime feature-classifier fit-classifier-naive-bayes \--i-reference-reads silva-132-99-515-806-nb-classifier.qza \--i-reference-taxonomy silva-132-99-515-806-nb-classifier.qza \--o-classifier classifier.qza#  使用训练好的分类器对样本数据进行分类
qiime feature-classifier classify-sklearn \--i-classifier classifier.qza \--i-reads paired-end-demux.qza \--o-classification taxonomy.qza

• q2-feature-table

q2-feature-table 插件是 QIIME 2 中的一个重要插件，用于处理和操作特征表（feature table）数据，它包括了许多功能，用于对微生物组数据进行分析和可视化。

以下是 q2-feature-table 插件的一些主要功能和使用方法：

1. 导入特征表数据： 可以使用该插件将不同格式的特征表数据导入到 QIIME 2 中，如BIOM格式、文本格式、或其他常见格式的特征表。

2. 特征表汇总和统计： 可以对特征表进行汇总和统计描述，比如计算每个样本中的特征数量、每个特征在样本中的出现频率等。

3. 特征表的过滤和修剪： 提供了多种方法对特征表进行过滤和修剪，如去除低频特征、去除低丰度特征、保留指定样本数或特征数等。

4. 特征表的转换和变换： 可以对特征表进行转换，如转置、归一化、对数转换等，以适应不同类型的分析需求。

5. 特征表的合并和拆分： 可以将多个特征表合并为一个，也可以根据样本元数据信息将特征表拆分为多个子集。

6. 特征表的可视化： 支持对特征表进行可视化展示，比如生成特征数量分布图、绘制热图展示特征在样本中的丰度等。

qiime feature-table --help
Usage: qiime feature-table [OPTIONS] COMMAND [ARGS]...Description: This is a QIIME 2 plugin supporting operations on sample byfeature tables, such as filtering, merging, and transforming tables.Plugin website: https://github.com/qiime2/q2-feature-tableGetting user support: Please post to the QIIME 2 forum for help with thisplugin: https://forum.qiime2.orgOptions:--version            Show the version and exit.--example-data PATH  Write example data and exit.--citations          Show citations and exit.--help               Show this message and exit.Commands:core-features                  Identify core features in tablefilter-features                Filter features from tablefilter-features-conditionally  Filter features from a table based onabundance and prevalencefilter-samples                 Filter samples from tablefilter-seqs                    Filter features from sequencesgroup                          Group samples or features by a metadatacolumnheatmap                        Generate a heatmap representation of afeature tablemerge                          Combine multiple tablesmerge-seqs                     Combine collections of feature sequencesmerge-taxa                     Combine collections of feature taxonomiespresence-absence               Convert to presence/absencerarefy                         Rarefy tablerelative-frequency             Convert to relative frequenciesrename-ids                     Renames sample or feature ids in a tablesplit                          Split one feature table into manysubsample                      Subsample tablesummarize                      Summarize tabletabulate-seqs                  View sequence associated with each featuretranspose                      Transpose a feature table.

#特征表的摘要和统计信息
qiime feature-table summarize \--i-table feature-table.qza \--o-visualization feature-table-summary.qzv#特征表的过滤和修剪
qiime feature-table filter-features \--i-table feature-table.qza \--p-min-frequency 10 \--o-filtered-table filtered-feature-table.qzaqiime feature-table filter-samples \--i-table feature-table.qza \--p-min-frequency 500 \--o-filtered-table filtered-sample-table.qza# 特征表的合并和操作
qiime feature-table merge \--i-tables table1.qza \--i-tables table2.qza \--o-merged-table merged-table.qza#计算特征表的β-diversity：
qiime diversity beta \--i-table feature-table.qza \--o-distance-matrix beta-diversity.qza \--p-metric braycurtis#可视化特征表
qiime feature-table summarize \--i-table feature-table.qza \--o-visualization feature-table-summary.qzv#可视化β-diversity距离矩阵
qiime diversity beta-phylogenetic \--i-table feature-table.qza \--i-phylogeny rooted-tree.qza \--o-distance-matrix beta-diversity.qza \--p-metric unweighted_unifracqiime diversity pcoa \--i-distance-matrix beta-diversity.qza \--o-pcoa pcoa-results.qzaqiime emperor plot \--i-pcoa pcoa-results.qza \--m-metadata-file sample-metadata.tsv \--o-visualization emperor.qzv

• q2-fragment-insertion

q2-fragment-insertion插件是用于将未分配的DNA序列（通常是16S rRNA或18S rRNA序列）嵌入（插入）到预先构建的参考进化树中的工具。这个插件可以帮助解决一些问题，比如通过将未知序列嵌入到进化树中，来推断未知序列的系统发育位置。

qiime fragment-insertion --help
Usage: qiime fragment-insertion [OPTIONS] COMMAND [ARGS]...Description: No description available. See plugin website:https://github.com/qiime2/q2-fragment-insertionPlugin website: https://github.com/qiime2/q2-fragment-insertionGetting user support: https://github.com/qiime2/q2-fragment-insertion/issuesOptions:--version            Show the version and exit.--example-data PATH  Write example data and exit.--citations          Show citations and exit.--help               Show this message and exit.Commands:classify-otus-experimental  Experimental: Obtain taxonomic lineages, byfinding closest OTU in reference phylogeny.filter-features             Filter fragments in tree from table.sepp                        Insert fragment sequences using SEPP intoreference phylogenies.

qiime fragment-insertion sepp \--i-representative-sequences <代表序列文件.qza> \--i-reference-database <参考数据库.qza> \--o-tree <输出进化树.qza> \--o-placements <输出插入位置文件.qza>

• --i-representative-sequences 是代表性序列文件的位置。
• --i-reference-database 是参考数据库文件的位置。
• --o-tree 指定输出的进化树文件。
• --o-placements 指定输出的插入位置文件。

• q2-longitudinal

q2-longitudinal 插件是 QIIME 2 中的一个插件，专门用于处理微生物组长期研究的数据。该插件允许用户对时间序列实验数据进行分析，以便检测微生物组随时间变化的情况，比较不同条件下的变化，以及对这些变化的统计显著性进行评估。

以下是 q2-longitudinal 插件的一些主要功能和使用命令：

主要功能：

1. 时间序列数据可视化：生成时间序列样本数据的可视化图表，比如长期研究的变化趋势、样本之间的差异等。
2. 差异分析：比较不同时间点或不同处理组之间的微生物组成差异。
3. Alpha 和 Beta 多样性分析：评估微生物群落在时间序列中的多样性和相似性变化。
4. 线性混合效应模型：对微生物组数据进行线性模型分析，以研究时间、处理效应和其交互作用对微生物组成的影响。

qiime longitudinal --help
Usage: qiime longitudinal [OPTIONS] COMMAND [ARGS]...Description: This QIIME 2 plugin supports methods for analysis of timeseries data, involving either paired sample comparisons or longitudinalstudy designs.Plugin website: https://github.com/qiime2/q2-longitudinalGetting user support: Please post to the QIIME 2 forum for help with thisplugin: https://forum.qiime2.orgOptions:--version            Show the version and exit.--example-data PATH  Write example data and exit.--citations          Show citations and exit.--help               Show this message and exit.Commands:anova                    ANOVA testfeature-volatility       Feature volatility analysisfirst-differences        Compute first differences or difference frombaseline between sequential statesfirst-distances          Compute first distances or distance from baselinebetween sequential stateslinear-mixed-effects     Linear mixed effects modelingmaturity-index           Microbial maturity index prediction.nmit                     Nonparametric microbial interdependence testpairwise-differences     Paired difference testing and boxplotspairwise-distances       Paired pairwise distance testing and boxplotsplot-feature-volatility  Plot longitudinal feature volatility andimportancesvolatility               Generate interactive volatility plot

#导入数据
qiime longitudinal feature-table-merge \--i-longitudinal-feature-tables feature-table.qza \--o-merged-table merged-table.qza#线性混合效应模型
qiime longitudinal linear-mixed-effects \--m-metadata-file sample-metadata.tsv \--m-metadata-file timepoints.tsv \--p-metric Shannon_index \--p-group-columns treatment \--p-state-column time \--p-individual-id-column subject \--o-visualization lme-results.qzv#可视化
qiime longitudinal volatility \--i-table merged-table.qza \--m-metadata-file sample-metadata.tsv \--p-state-column time \--p-individual-id-column subject \--o-visualization volatility.qzv#组间差异
qiime longitudinal pairwise-differences \--m-metadata-file sample-metadata.tsv \--m-metadata-file timepoints.tsv \--p-metric Shannon_index \--p-group-column treatment \--p-state-column time \--p-individual-id-column subject \--o-visualization pairwise-differences.qzv#α多样性图表
qiime longitudinal maturity-index \--i-alpha-diversity alpha-diversity.qza \--m-metadata-file sample-metadata.tsv \--p-state-column time \--p-individual-id-column subject \--o-visualization maturity-index.qzv

• q2-metadata

q2-metadata 插件用于处理和操作元数据，元数据是描述样本信息的数据，比如样本来源、处理方法、实验条件等。以下是 q2-metadata 插件的简要介绍和一些常见的使用命令：

q2-metadata 插件的功能：

1. 元数据导入： 将元数据文件导入到 QIIME 2 格式中。
2. 元数据可视化： 可视化元数据内容以便更好地理解样本信息。
3. 元数据的处理和编辑： 对元数据进行筛选、编辑和转换。
4. 元数据统计和摘要： 统计和生成关于元数据的摘要信息。

qiime metadata --help
Usage: qiime metadata [OPTIONS] COMMAND [ARGS]...Description: This QIIME 2 plugin provides functionality for working with andvisualizing Metadata.Plugin website: https://github.com/qiime2/q2-metadataGetting user support: Please post to the QIIME 2 forum for help with thisplugin: https://forum.qiime2.orgOptions:--version            Show the version and exit.--example-data PATH  Write example data and exit.--citations          Show citations and exit.--help               Show this message and exit.Commands:distance-matrix  Create a distance matrix from a numeric Metadata columnmerge            Merge metadatashuffle-groups   Shuffle values in a categorical sample metadata column.tabulate         Interactively explore Metadata in an HTML table

# 1. 元数据导入：
qiime metadata tabulate --m-input-file sample-metadata.tsv --o-visualization sample-metadata.qzv
# 2. 元数据可视化：
qiime metadata tabulate --m-input-file sample-metadata.qza --o-visualization sample-metadata.qzv
# 3. 元数据编辑与处理：
#  删除列：
qiime metadata tabulate --m-input-file sample-metadata.qza --o-visualization sample-metadata.qzv
#  筛选行（过滤）：
qiime metadata tabulate --m-input-file sample-metadata.qza --o-visualization sample-metadata.qzv
# 4. 元数据统计和摘要：
qiime metadata tabulate --m-input-file sample-metadata.qza --o-visualization sample-me

• q2-phylogeny

q2-phylogeny插件则提供了处理生物多样性分析中的系统发育信息的功能。该插件主要用于构建系统发育树和处理系统发育树相关的操作，例如序列的进化树推断、进化树的根节点分配等。

qiime phylogeny --help
Usage: qiime phylogeny [OPTIONS] COMMAND [ARGS]...Description: This QIIME 2 plugin supports generating and manipulatingphylogenetic trees.Plugin website: https://github.com/qiime2/q2-phylogenyGetting user support: Please post to the QIIME 2 forum for help with thisplugin: https://forum.qiime2.orgOptions:--version            Show the version and exit.--example-data PATH  Write example data and exit.--citations          Show citations and exit.--help               Show this message and exit.Commands:align-to-tree-mafft-fasttree  Build a phylogenetic tree using fasttree andmafft alignmentalign-to-tree-mafft-iqtree    Build a phylogenetic tree using iqtree andmafft alignment.align-to-tree-mafft-raxml     Build a phylogenetic tree using raxml andmafft alignment.fasttree                      Construct a phylogenetic tree with FastTree.filter-table                  Remove features from table if they're notpresent in tree.filter-tree                   Remove features from tree based on metadataiqtree                        Construct a phylogenetic tree with IQ-TREE.iqtree-ultrafast-bootstrap    Construct a phylogenetic tree with IQ-TREEwith bootstrap supports.midpoint-root                 Midpoint root an unrooted phylogenetic tree.raxml                         Construct a phylogenetic tree with RAxML.raxml-rapid-bootstrap         Construct a phylogenetic tree with bootstrapsupports using RAxML.robinson-foulds               Calculate Robinson-Foulds distance betweenphylogenetic trees.

## 构建系统发育树：
#使用多序列比对后的结果构建系统发育树。可以使用q2-phylogeny中的FastTree或RAxML进行树的构建。以下是使用FastTree的示例命令：
qiime phylogeny fasttree \--i-alignment aligned-sequences.qza \--o-tree tree.qza# 或者使用RAxML进行系统发育树的构建：
qiime phylogeny raxml \--i-alignment aligned-sequences.qza \--p-substitution-model GTRGAMMA \--o-tree tree.qza \--verbose## 可选操作 - 根节点分配：
# 有时，你可能需要为系统发育树分配根节点。可以使用q2-phylogeny中的根节点分配插件进行此操作。以下是一个示例命令：
qiime phylogeny midpoint-root \--i-tree tree.qza \--o-rooted-tree rooted-tree.qza

• q2-quality-control

q2-quality-control 插件旨在进行序列数据的质量控制和过滤，它可以执行以下任务：

• 对序列数据进行质量评估
• 去除低质量序列
• 截取或修剪序列的部分
• 去除嵌合序列（chimeras）
• 过滤低频序列

• q2-quality-filter

q2-quality-filter插件用于对DNA测序数据进行质量控制和过滤，以去除低质量的序列。这有助于提高后续分析的准确性和可靠性。

qiime quality-control --help
Usage: qiime quality-control [OPTIONS] COMMAND [ARGS]...Description: This QIIME 2 plugin supports methods for assessing andcontrolling the quality of feature and sequence data.Plugin website: https://github.com/qiime2/q2-quality-controlGetting user support: Please post to the QIIME 2 forum for help with thisplugin: https://forum.qiime2.orgOptions:--version            Show the version and exit.--example-data PATH  Write example data and exit.--citations          Show citations and exit.--help               Show this message and exit.Commands:bowtie2-build              Build bowtie2 index from reference sequences.decontam-identify          Identify contaminantsdecontam-identify-batches  Identify contaminants in Batch Modedecontam-remove            Removes contaminantdecontam-score-viz         Generate a histogram representation of the scoresevaluate-composition       Evaluate expected vs. observed taxonomiccomposition of samplesevaluate-seqs              Compare query (observed) vs. reference (expected)sequences.evaluate-taxonomy          Evaluate expected vs. observed taxonomicassignmentsexclude-seqs               Exclude sequences by alignmentfilter-reads               Filter demultiplexed sequences by alignment toreference database.

qiime quality-filter q-score \--i-demux paired-end-demux.qza \--p-min-quality 20 \--o-filtered-sequences demux-filtered.qza \--o-filter-stats demux-filter-stats.qza

• q2-sample-classifier

而q2-sample-classifier是Qiime 2中的一个插件，用于样本分类和预测。它可以帮助用户利用机器学习算法对样本进行分类，比较不同条件下的微生物组成差异，例如，预测分类数据（如临床数据）和微生物组成之间的关系。

qiime sample-classifier --help
Usage: qiime sample-classifier [OPTIONS] COMMAND [ARGS]...Description: This QIIME 2 plugin supports methods for supervisedclassification and regression of sample metadata, and other supervisedmachine learning methods.Plugin website: https://github.com/qiime2/q2-sample-classifierGetting user support: Please post to the QIIME 2 forum for help with thisplugin: https://forum.qiime2.orgOptions:--version            Show the version and exit.--example-data PATH  Write example data and exit.--citations          Show citations and exit.--help               Show this message and exit.Commands:classify-samples            Train and test a cross-validated supervisedlearning classifier.classify-samples-from-dist  Run k-nearest-neighbors on a labeled distancematrix.classify-samples-ncv        Nested cross-validated supervised learningclassifier.confusion-matrix            Make a confusion matrix from sample classifierpredictions.fit-classifier              Fit a supervised learning classifier.fit-regressor               Fit a supervised learning regressor.heatmap                     Generate heatmap of important features.metatable                   Convert (and merge) positive numeric metadata(in)to feature table.predict-classification      Use trained classifier to predict target valuesfor new samples.predict-regression          Use trained regressor to predict target valuesfor new samples.regress-samples             Train and test a cross-validated supervisedlearning regressor.regress-samples-ncv         Nested cross-validated supervised learningregressor.scatterplot                 Make 2D scatterplot and linear regression ofregressor predictions.split-table                 Split a feature table into training and testingsets.summarize                   Summarize parameter and feature extractioninformation for a trained estimator.

qiime sample-classifier classify-samples \--i-table feature-table.qza \--m-metadata-file sample-metadata.qza \--m-metadata-column TARGET_COLUMN \--p-test-size 0.2 \--p-random-state 42 \--p-n-estimators 100 \--p-n-jobs 1 \--o-visualization classification-results.qzv

• --i-table 指定特征表格的位置
• --m-metadata-file 指定样本元数据的位置
• --m-metadata-column 指定用于分类的目标列
• --p-test-size 设置测试集的比例
• --p-random-state 设置随机种子以确保结果可重复
• --p-n-estimators 设置分类器使用的估计器数量
• --p-n-jobs 设置用于计算的作业数
• --o-visualization 指定输出结果的位置

• q2-taxa

q2-taxa 插件则用于分析和可视化分类学信息，特别是对于已经进行了序列分类（比如16S rRNA）的数据。q2-taxa 允许用户对物种注释信息进行处理、汇总和可视化。

qiime taxa --help
Usage: qiime taxa [OPTIONS] COMMAND [ARGS]...Description: This QIIME 2 plugin provides functionality for working with andvisualizing taxonomic annotations of features.Plugin website: https://github.com/qiime2/q2-taxaGetting user support: Please post to the QIIME 2 forum for help with thisplugin: https://forum.qiime2.orgOptions:--version            Show the version and exit.--example-data PATH  Write example data and exit.--citations          Show citations and exit.--help               Show this message and exit.Commands:barplot       Visualize taxonomy with an interactive bar plotcollapse      Collapse features by their taxonomy at the specified levelfilter-seqs   Taxonomy-based feature sequence filter.filter-table  Taxonomy-based feature table filter.

# 1. 导入数据
# 使用命令 qiime taxa 可以导入并生成用于分类学分析的数据格式。例如：
qiime taxa import \--input-path taxonomy.tsv \--output-path taxonomy.qza \--type 'FeatureData[Taxonomy]'# 2. 查看物种丰度
# 命令 qiime taxa barplot 可以用于生成物种丰度的柱状图，用于可视化各个样本中不同分类水平的相对丰度。
qiime taxa barplot \--i-table table.qza \--i-taxonomy taxonomy.qza \--m-metadata-file sample-metadata.tsv \--o-visualization taxa-bar-plot.qzv# 3. 物种注释
# 可通过 qiime taxa classify-sklearn 命令使用机器学习分类器进行序列的物种注释。以下是一个例子：
qiime taxa classify-sklearn \--i-reads rep-seqs.qza \--i-classifier classifier.qza \--o-classification taxonomy.qza# 4. 特定分类水平的筛选和可视化
# 可以使用 qiime taxa filter-table 和 qiime taxa filter-seqs 命令在特定分类水平对数据进行过滤。然后，可以使用其他命令可视化已过滤的数据。# 5. 物种多样性分析
使用 qiime taxa collapse 和 qiime taxa rarefy 等命令可以对物种注释数据进行聚合和稀释，以进行多样性分析。

• q2-types

q2-types插件旨在处理和管理不同类型的数据，包括：

• DNA 序列
• RNA 序列
• 蛋白质序列
• 样本和元数据信息

• q2-vsearch

 q2-vsearch 插件则是 QIIME 2 中用于序列相似性搜索和聚类的插件之一。它基于 VSEARCH，提供了执行序列相似性搜索、聚类和序列分析的功能。这个基本上与直接使用vesearch直接使用差不多了，只不过这里集成后可以直接与qiime的其他模块相结合使用。

qiime vsearch --help
Usage: qiime vsearch [OPTIONS] COMMAND [ARGS]...Description: This plugin wraps the vsearch application, and provides methodsfor clustering and dereplicating features and sequences.Plugin website: https://github.com/qiime2/q2-vsearchGetting user support: Please post to the QIIME 2 forum for help with thisplugin: https://forum.qiime2.orgOptions:--version            Show the version and exit.--example-data PATH  Write example data and exit.--citations          Show citations and exit.--help               Show this message and exit.Commands:cluster-features-closed-referenceClosed-reference clustering of features.cluster-features-de-novo        De novo clustering of features.cluster-features-open-referenceOpen-reference clustering of features.dereplicate-sequences           Dereplicate sequences.fastq-stats                     Fastq stats with vsearch.merge-pairs                     Merge paired-end reads.uchime-denovo                   De novo chimera filtering with vsearch.uchime-ref                      Reference-based chimera filtering with

# 序列相似性搜索
# 使用 q2-vsearch 插件进行序列相似性搜索的命令：
qiime vsearch search-sequences \--i-query query_sequences.qza \--i-reference reference_sequences.qza \--o-results search_results.qza \--o-perc-identity output_identity.qza \--o-failed-sequences failed_sequences.qza
# 此命令将执行一项序列搜索，将查询序列与参考序列集进行比较，并生成包含搜索结果的文件。# 序列聚类
# 利用 q2-vsearch 进行序列聚类的命令示例：
qiime vsearch cluster-features-de-novo \--i-sequences sequences.qza \--i-table table.qza \--p-perc-identity 0.97 \--o-clustered-table clustered_table.qza \--o-clustered-sequences clustered_sequences.qza \--o-new-reference-sequences new_reference_sequences.qza

当然还有分析结果的可视化

在线可视化，拖拽出图：QIIME 2 View