QIIME2微生物组分析流程

• QIIME2功能介绍

Usage: qiime [OPTIONS] COMMAND [ARGS]...
Commands:
  info                Display information about current deployment.
  tools               Tools for working with QIIME 2 files.
  dev                 Utilities for developers and advanced users.
  alignment           Plugin for generating and manipulating alignments.
  composition         Plugin for compositional data analysis.
  cutadapt            Plugin for removing adapter sequences, primers, and other unwanted sequence from sequence data.
  dada2               Plugin for sequence quality control with DADA2.
  deblur              Plugin for sequence quality control with Deblur.
  demux               Plugin for demultiplexing & viewing sequence quality.
  diversity           Plugin for exploring community diversity.
  emperor             Plugin for ordination plotting with Emperor.
  feature-classifier  Plugin for taxonomic classification.
  feature-table       Plugin for working with sample by feature tables.
  fragment-insertion  Plugin for extending phylogenies.
  gneiss              Plugin for building compositional models.
  longitudinal        Plugin for paired sample and time series analyses.
  metadata            Plugin for working with Metadata.
  phylogeny           Plugin for generating and manipulating phylogenies.
  quality-control     Plugin for quality control of feature and sequence data.
  quality-filter      Plugin for PHRED-based filtering and trimming.
  sample-classifier   Plugin for machine learning prediction of sample metadata.
  taxa                Plugin for working with feature taxonomy annotations.
  vsearch             Plugin for clustering and dereplicating with vsearch.

1、数据导入

Usage: qiime tools import [OPTIONS]
Options:
  --type TEXT             每步分析都会产生qza文件，都会有相应的语义类型，避免用户不合理的分析过程。
  --input-path PATH       输入文件路径，文件格式为sample-id,absolute-filepath,direction
  --output-path ARTIFACT  输出文件：.qza格式的文件名
  --input-format TEXT     输入数据格式：双端测序数据可设置为PairedEndFastqManifestPhred33V2
  --show-importable-types   查看所有的语义类型
  --show-importable-formats 查看所有输入数据格式
  --help                    查看说明

• 示例

# 导入原始序列
qiime tools import \ 
--type 'SampleData[PairedEndSequencesWithQuality]' \   #成对的带质量序列，要求序列ID与样品编号存在对应关系
--input-path sample_list.txt \ 
--input-format PairedEndFastqManifestPhred33 \ 
--output-path demux-paired-end.qza 
# 导入.biom格式的特征表：转换成qza格式
qiime tools import \ 
--type 'FeatureTable[Frequency]' \     # FeatureTable[Frequency]: 频率，即Feature表(OTU表)，为每个样品中对应OTU出现频率的表格
--input-path feature_table.biom \ 
--input-format BIOMV210Format  \ 
--output-path feature_table.qza

2、生成OUT表

Usage: qiime dada2 [OPTIONS] COMMAND [ARGS]...
主要作用是去除低质量序列、嵌合体；再生成OTU表，现在叫Feature表，因为不再使用聚类方法，相当于QIIME时代100%相似度的OTU表。
Options:
  --version    Show the version and exit.
  --citations  Show citations and exit.
  --help       Show this message and exit.
Commands:
  denoise-paired  Denoise and dereplicate paired-end sequences
  denoise-pyro    Denoise and dereplicate single-end pyrosequences
  denoise-single  Denoise and dereplicate single-end sequences

• 示例

qiime dada2 denoise-paired \ 
--i-demultiplexed-seqs demux-paired-end.qza \ 
--o-table feature_table.qza \ 
--o-representative-sequences rep-seqs-dada2.qza \   #输出代表性序列文件
--o-denoising-stats stats-dada2.qza \   #过程统计
--p-trim-left-f 0 \      #表示从forward左端的第0位置开始
--p-trim-left-r 0 \      #表示从reverse左端的第0位置开始
--p-trunc-len-f 250 \    #表示forward总共保留的长度为250
--p-trunc-len-r 250     #表示reverse总共保留的长度为250 

3、操作特征表

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.
Commands:
  core-features       Identify core features in table
  filter-features     Filter features from table
  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
  subsample           Subsample table
  summarize           Summarize table
  tabulate-seqs       View sequence associated with each feature
  transpose           Transpose a feature table.

• 特征表统计

qiime feature-table summarize \ 
--i-table feature_table.qza \ 
--o-visualization feature_table.qzv
# QIIME2生成的图表结果文件类型，以.qzv为扩展名，末尾的v代表visual

• 过滤特征表：过滤样本

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

4、多样性分析

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.
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
  pcoa                       Principal Coordinate Analysis
  pcoa-biplot                Principal Coordinate Analysis Biplot
  procrustes-analysis        Procrustes Analysis 

• 核心多样性（无系统发育树）

qiime diversity core-metrics \ 
--i-table feature_table.qza \ 
--p-sampling-depth 138000 \ 
--m-metadata-file sample_metadata.tsv \ 
--output-dir /data/hushy/XJ/data_BFV/qiime2/diversity  #自动创建目录
# alpha多样性observed_otus(OTU数量)指数 observed_otus_vector.qza
# alpha多样性香农熵(考虑物种和丰度)指数 shannon_vector.qza
# alpha多样性evenness(均匀度，考虑物种和丰度)指数 evenness_vector.qza
# beta多样性bray_curtis距离矩阵 bray_curtis_distance_matrix.qza
# beta多样性可视化结果bray_curtis_emperor.qzv，图中Axis1 表示尽可能最大解释数据变化的主坐标成分，Axis2 为解释余下的变化度中占比例最大的主坐标成分，Axis3 等依次类推。
#Richness, Chao1，Shannon三个指数是常用的评估丰富度的指标，数值越高表明样品包含的物种丰富度就越高。
#Jaccard距离（群落差异的定性度量，即只考虑种类，不考虑丰度）
#Bray-Curtis距离（群落差异的定量度量）
#非加权UniFrac距离（包含特征之间的系统发育关系的群落差异定性度量）
#加权UniFrac距离（包含特征之间的系统发育关系的群落差异定量度量）

• alpha多样性可视化及组间显著性分析

qiime diversity alpha-group-significance \ 
--i-alpha-diversity shannon_vector.qza \ 
--m-metadata-file sample_metadata.tsv \ 
--o-visualization shannon_vector.qzv