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 --help
Usage: 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 semantic
types that can be imported.
Options:
--type TEXT The semantic type of the artifact that will be
created upon importing. Use --show-importable-types
to see what importable semantic types are available
in 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 not
provided, data must be in the format expected by the
semantic 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-formats
Show formats that can be supplied to --input-format
to 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-sequences
qiime 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 --importable2、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.html
qiime 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.ReferenceOTU1212:0.00521
mkdir 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.qzv
qiime metadata tabulate \
--m-input-file faith_pd_vector.qza \
--o-visualization tabulated-faith-pd-metadata.qzv
qiime metadata tabulate \
--m-input-file sample-metadata.tsv \
--m-input-file faith_pd_vector.qza \
--o-visualization tabulated-combined-metadata.qzv
qiime 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.qzaFiltering 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.qza
qiime 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.qza5、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：
- 序列比对：对DNA、RNA或蛋白质序列进行比对。
- 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。
- 生成比对结果：产生比对结果，以便后续分析或可视化。
- 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 and
manipulating sequence alignments.
Plugin website: https://github.com/qiime2/q2-alignment
Getting user support: Please post to the QIIME 2 forum for help with this
plugin: https://forum.qiime2.org
Options:
--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 MAFFT
mafft-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 data
analysis.
Plugin website: https://github.com/qiime2/q2-composition
Getting user support: Please post to the QIIME 2 forum for help with this
plugin: https://forum.qiime2.org
Options:
--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 Correction
da-barplot Differential abundance bar plots
tabulate 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.qza
qiime 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-cutadapt
Getting user support: Please post to the QIIME 2 forum for help with this
plugin: https://forum.qiime2.org
Options:
--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-end
sequences.
trim-single Find and remove adapters in demultiplexed single-end
sequences.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 quality
control 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 this
plugin: https://forum.qiime2.org
Options:
--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 CCS
denoise-paired Denoise and dereplicate paired-end sequences
denoise-pyro Denoise and dereplicate single-end pyrosequences
denoise-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：
- 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。
- 质量过滤： 过滤低质量序列，去除低质量序列读数。
- 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。
- 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 performing
sequence quality control.
Plugin website: https://github.com/biocore/deblur
Getting user support: Please post to the QIIME 2 forum for help with this
plugin: https://forum.qiime2.org
Options:
--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：
-
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。
-
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 and
paired-end sequence reads and visualization of sequence quality information.
Plugin website: https://github.com/qiime2/q2-demux
Getting user support: Please post to the QIIME 2 forum for help with this
plugin: https://forum.qiime2.org
Options:
--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 generated
with the EMP protocol.
emp-single Demultiplex sequence data generated with the EMP
protocol.
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 without
replacement.
subsample-single Subsample single-end sequences without
replacement.
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 and
exploring community alpha and beta diversity through statistics and
visualizations in the context of sample metadata.
Plugin website: https://github.com/qiime2/q2-diversity
Getting user support: Please post to the QIIME 2 forum for help with this
plugin: https://forum.qiime2.org
Options:
--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 significance
alpha Alpha diversity
alpha-correlation Alpha diversity correlation
alpha-group-significance Alpha diversity comparisons
alpha-phylogenetic Alpha diversity (phylogenetic)
alpha-rarefaction Alpha rarefaction curves
beta Beta diversity
beta-correlation Beta diversity correlation
beta-group-significance Beta diversity group significance
beta-phylogenetic Beta diversity (phylogenetic)
beta-rarefaction Beta diversity rarefaction
bioenv bioenv
core-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 matrices
partial-procrustes Partial Procrustes
pcoa Principal Coordinate Analysis
pcoa-biplot Principal Coordinate Analysis Biplot
procrustes-analysis Procrustes Analysis
tsne t-distributed stochastic neighbor embedding
umap 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
- 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。
- 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。
- 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。
- 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 community
alpha and beta diversity.
Plugin website: https://github.com/qiime2/q2-diversity-lib
Getting user support: Please post to the QIIME 2 forum for help with this
plugin: https://forum.qiime2.org
Options:
--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-passthrough
Beta Phylogenetic Meta Passthrough
beta-phylogenetic-passthrough Beta Phylogenetic Passthrough
bray-curtis Bray-Curtis Dissimilarity
faith-pd Faith's Phylogenetic Diversity
jaccard Jaccard Distance
observed-features Observed Features
pielou-evenness Pielou's Evenness
shannon-entropy Shannon's Entropy
unweighted-unifrac Unweighted Unifrac
weighted-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 interactive
visualization of ordination plots.
Plugin website: http://emperor.microbio.me
Getting user support: Please post to the QIIME 2 forum for help with this
plugin: https://forum.qiime2.org
Options:
--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 Analysis
Biplot
plot Visualize and Interact with Principal Coordinates Analysis
Plots
procrustes-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 of
features using a variety of methods, including Naive Bayes, vsearch, and
BLAST+.
Plugin website: https://github.com/qiime2/q2-feature-classifier
Getting user support: Please post to the QIIME 2 forum for help with this
plugin: https://forum.qiime2.org
Options:
--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 classifier
classify-consensus-vsearch VSEARCH-based consensus taxonomy classifier
classify-hybrid-vsearch-sklearn
ALPHA Hybrid classifier: VSEARCH exact match
+ sklearn classifier
classify-sklearn Pre-fitted sklearn-based taxonomy classifier
extract-reads Extract reads from reference sequences.
find-consensus-annotation Find consensus among multiple annotations.
fit-classifier-naive-bayes Train the naive_bayes classifier
fit-classifier-sklearn Train an almost arbitrary scikit-learn
classifier
makeblastdb 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：
-
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。
-
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。
-
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。
-
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。
-
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。
-
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 by
feature tables, such as filtering, merging, and transforming tables.
Plugin website: https://github.com/qiime2/q2-feature-table
Getting user support: Please post to the QIIME 2 forum for help with this
plugin: https://forum.qiime2.org
Options:
--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 table
filter-features Filter features from table
filter-features-conditionally Filter features from a table based on
abundance and prevalence
filter-samples Filter samples from table
filter-seqs Filter features from sequences
group Group samples or features by a metadata
column
heatmap Generate a heatmap representation of a
feature table
merge Combine multiple tables
merge-seqs Combine collections of feature sequences
merge-taxa Combine collections of feature taxonomies
presence-absence Convert to presence/absence
rarefy Rarefy table
relative-frequency Convert to relative frequencies
rename-ids Renames sample or feature ids in a table
split Split one feature table into many
subsample Subsample table
summarize Summarize table
tabulate-seqs View sequence associated with each feature
transpose 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.qza
qiime 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_unifrac
qiime diversity pcoa \
--i-distance-matrix beta-diversity.qza \
--o-pcoa pcoa-results.qza
qiime 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-insertion
Plugin website: https://github.com/qiime2/q2-fragment-insertion
Getting user support: https://github.com/qiime2/q2-fragment-insertion/issues
Options:
--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, by
finding closest OTU in reference phylogeny.
filter-features Filter fragments in tree from table.
sepp Insert fragment sequences using SEPP into
reference 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：
- 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。
- 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。
- 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。
- 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 time
series data, involving either paired sample comparisons or longitudinal
study designs.
Plugin website: https://github.com/qiime2/q2-longitudinal
Getting user support: Please post to the QIIME 2 forum for help with this
plugin: https://forum.qiime2.org
Options:
--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 test
feature-volatility Feature volatility analysis
first-differences Compute first differences or difference from
baseline between sequential states
first-distances Compute first distances or distance from baseline
between sequential states
linear-mixed-effects Linear mixed effects modeling
maturity-index Microbial maturity index prediction.
nmit Nonparametric microbial interdependence test
pairwise-differences Paired difference testing and boxplots
pairwise-distances Paired pairwise distance testing and boxplots
plot-feature-volatility Plot longitudinal feature volatility and
importances
volatility 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：
- 元数据导入： 将元数据文件导入到 QIIME 2 格式中。
- 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。
- 元数据的处理和编辑： 对元数据进行筛选、编辑和转换。
- 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 and
visualizing Metadata.
Plugin website: https://github.com/qiime2/q2-metadata
Getting user support: Please post to the QIIME 2 forum for help with this
plugin: https://forum.qiime2.org
Options:
--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 column
merge Merge metadata
shuffle-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 manipulating
phylogenetic trees.
Plugin website: https://github.com/qiime2/q2-phylogeny
Getting user support: Please post to the QIIME 2 forum for help with this
plugin: https://forum.qiime2.org
Options:
--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 and
mafft alignment
align-to-tree-mafft-iqtree Build a phylogenetic tree using iqtree and
mafft alignment.
align-to-tree-mafft-raxml Build a phylogenetic tree using raxml and
mafft alignment.
fasttree Construct a phylogenetic tree with FastTree.
filter-table Remove features from table if they're not
present in tree.
filter-tree Remove features from tree based on metadata
iqtree Construct a phylogenetic tree with IQ-TREE.
iqtree-ultrafast-bootstrap Construct a phylogenetic tree with IQ-TREE
with 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 bootstrap
supports using RAxML.
robinson-foulds Calculate Robinson-Foulds distance between
phylogenetic 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 and
controlling the quality of feature and sequence data.
Plugin website: https://github.com/qiime2/q2-quality-control
Getting user support: Please post to the QIIME 2 forum for help with this
plugin: https://forum.qiime2.org
Options:
--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 contaminants
decontam-identify-batches Identify contaminants in Batch Mode
decontam-remove Removes contaminant
decontam-score-viz Generate a histogram representation of the scores
evaluate-composition Evaluate expected vs. observed taxonomic
composition of samples
evaluate-seqs Compare query (observed) vs. reference (expected)
sequences.
evaluate-taxonomy Evaluate expected vs. observed taxonomic
assignments
exclude-seqs Exclude sequences by alignment
filter-reads Filter demultiplexed sequences by alignment to
reference 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 supervised
classification and regression of sample metadata, and other supervised
machine learning methods.
Plugin website: https://github.com/qiime2/q2-sample-classifier
Getting user support: Please post to the QIIME 2 forum for help with this
plugin: https://forum.qiime2.org
Options:
--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 supervised
learning classifier.
classify-samples-from-dist Run k-nearest-neighbors on a labeled distance
matrix.
classify-samples-ncv Nested cross-validated supervised learning
classifier.
confusion-matrix Make a confusion matrix from sample classifier
predictions.
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 values
for new samples.
predict-regression Use trained regressor to predict target values
for new samples.
regress-samples Train and test a cross-validated supervised
learning regressor.
regress-samples-ncv Nested cross-validated supervised learning
regressor.
scatterplot Make 2D scatterplot and linear regression of
regressor predictions.
split-table Split a feature table into training and testing
sets.
summarize Summarize parameter and feature extraction
information 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 and
visualizing taxonomic annotations of features.
Plugin website: https://github.com/qiime2/q2-taxa
Getting user support: Please post to the QIIME 2 forum for help with this
plugin: https://forum.qiime2.org
Options:
--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 plot
collapse Collapse features by their taxonomy at the specified level
filter-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 methods
for clustering and dereplicating features and sequences.
Plugin website: https://github.com/qiime2/q2-vsearch
Getting user support: Please post to the QIIME 2 forum for help with this
plugin: https://forum.qiime2.org
Options:
--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-reference
Closed-reference clustering of features.
cluster-features-de-novo De novo clustering of features.
cluster-features-open-reference
Open-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
