网站商业授权含义,建设网站书籍pdf下载,网站品牌推广公司,vs2012解决方案做网站QIIME 2 2023.9 Amplicon Distribution介绍#xff1a;
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#xff0c;包括了主要和常见的扩增子分析模块#xff0c;用户可以单独使用各个模块#xff0c;也可以使用各模块组成不同的分析流程。从2023.09版本…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metadataMultiplexed 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
ATGCAGCTCAGTIIIIIIIIIIIH
HWI-EAS440_0386:1:23:14818:1533#0/1
CCCCTCAGCGGCDDDD?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
TACGNAGGATCCGAGCGTTATCCGGATTTATTGGGTTTAAAGGGAGCGTAGATGGATGTTTAAGTCAGTTGTGAAAGTTTGCGGCTCAACCGTAAAATTGCAGTTGATACTGGATATCTTGAGTGCAGTTGAGGCAGGGGGGGATTGGTGTGIIIE)EEEEEEEEGFIIGIIIHIHHGIIIGIIHHHGIIHGHEGDGIFIGEHGIHHGHHGHHGGHEEGHEGGEHEBBHBBEEDCEDDDB?BEBCBABA??08;3;8:5;6?######################
HWI-EAS440_0386:1:23:14818:1533#0/1
CCCCNCAGCGGCAAAAATTAAAATTTTTACCGCTTCGGCGTTATAGCCTCACACTCAATCTTTTATCACGAAGTCATGATTGAATCGCGAGTGGTCGGCAGATTGCGATAAACGGGCACATTAAATTTAAACTGATGATTCCACTGCAACAA642$24;1)/:*B?BBDDBBDBDD############################################################################################################################
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#AAFACAA4BGCEEECGGHGGEFCFBG#BA###BABAEFGEEE##BBAA###########B######################################################################################
M00176:65:000000000-A41FR:1:1101:16939:1420 1:N:0:0
NACGTAGGGGGCAAGCGTTGTCCGGAATCATTGGNNGTAAAGAGCGTGNAGGCNNNNNGNNANNTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN#ABAFABBBGGGGEEFGGGHGGFFGFHHHHH##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######################################################################################?#?##?#?###########??/?FCFBF#/??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-8ABCC5811884::99?EECFFGDFADECFFFEEDDEFEDEDFFFEEFCCBCFCCB3CFF:BBFFFCCD,89CC:5:ACFDCFBEGFFFDGCCEC?FGGGGGGGGGCFGGFCFGGGGGGGGGGEG7CFFGFFFGGGFG?FACE;:8CCCCEEF9FFFEGGC**/:3:2CC:C;C81;C9?FF8C758CGGG2:7DCEECEFE927CF492/8B7D)7FFFCFF*9F522,2890:44AB49(3F51).69D?D34*44:45B?::086
M00899:113:000000000-A5K20:1:1101:25454:3578 1:N:0:2
CCTACGGGAGGCAGCAGTGAGGAATATTGGTCAATGGGCGAGAGCCTGAACCAGCCAAGTAGCGTGCAGGATGACGGCCCTATGGGTTGTAAACTGCTTTTGTATGGGGATAAAGTCAGTCACGTGTGATTGTTTGCAGGTACCATACGAATAAGGACCGGCTAATTCCGTGCCAGCAGCCGCGGTAATACGGAAGGTCCGGGCGTTATCCGGATTTATTGGGTTTAAAGGGAGCGTAGGCTGGAGATTAAGTGTGTTGTGAAATGTAGACGCTCAACGTCTGACTTGCAGCGCATACTGG8ACCCGDAA18;CEFGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGFGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGEGGGGGGFFF?FGGGGGGGGEGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGCGFGGGFFFGGGGGGEFGGGGGGGGGGGGCGEGGDGGGGGGGGGGCGGCECDGGFGGGGGGGGFGGGFCBFFEGFFFFF:FGGF66:AFBGFFFFFFA9AAFB?0)C:0CF?C46FAD??90;::?DA
M00899:113:000000000-A5K20:1:1101:25177:3605 1:N:0:2
CCTACGGGAGGCAGCAGTGAGGAATATTGGTCAATGGACGGAAGTCTGAACCAGCCAAGTAGCGTGCAGGATGACGGCCCTATGGGTTGTAAACTGCTTTTGTATGGGGATAAAGTTAGGGACGTGTCCCTATTTGCAGGTACCATACGAATAAGGACCGGCTAATTCCGTGCCAGCAGCCGCGGTAATACGGAAGGTCCAGGCGTTATCCGGATTTATTGGGTTTAAAGGGAGCGTAGGCTGGAGATTAAGTGTGTTGTGAAATGTAGACGCTCAACGTCTGAATTGCAGCGCATACTGG88BCCEDAD9018;;CCFGGGGFGGGFGGGGGGGGGGGGGGGGGGGGGGGFGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGFGGGGGGGGGGGGGFGGGGGGGGGGGGGGGGEGDGGGGGGGGGFFGGGGGGGGGFGGGFGGGFGGGFFGCGGGGGGFGGFDGGGGGGGGGGGGG5CBFGCGGGGC?FGGGGGGGGGGGDEGDDDGFGGGGGEGGGGGGA39BFFDDEF4:D5CE?CFFFABGFFF9A246B::DE?FGGFG4F?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.qzawget \-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?spm1001.2014.3001.5503
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
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 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.qzvq2-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 sequencesqiime 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 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_statsq2-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 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.qzvq2-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
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PCoAPrincipal 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PERMANOVAPermutational 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 Faiths Phylogenetic Diversityjaccard Jaccard Distanceobserved-features Observed Featurespielou-evenness Pielous Evennessshannon-entropy Shannons 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 permanovaq2-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 plotq2-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.qzaq2-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 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.qzvq2-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
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 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.qzvq2-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 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 theyre 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.qzaq2-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
