0046-【宏基因组】-qiime2官方教程实践1-Moving pictures of the human microbiome

1. 数据文章——Moving pictures of the human microbiome

文章下载：https://www.ncbi.nlm.nih.gov/pubmed/21624126

样本选取：
2个生物个体，4个部分，396个时间点

文库构建：
Meta16S V4区文库

测序策略：
illunima 、454
不同区域，选择不同的二代测序平台

信息分析：
qiime2-2018.4 软件，及测试代码集
注：与中文的帮助文档2017.7代码有少量不同

本次测试：
本示例的的数据来自文章《Moving pictures of the human microbiome》，Genome Biology 2011，取样来自两个人身体四个部位五个时间点。

2. 流程实践

1.准备数据

# 下载实验设计表
wget http://bailab.genetics.ac.cn/markdown/sample-metadata.tsv

# 下载实验测序数据
mkdir -p emp-single-end-sequences
wget -O "emp-single-end-sequences/barcodes.fastq.gz" "https://data.qiime2.org/2017.7/tutorials/moving-pictures/emp-single-end-sequences/barcodes.fastq.gz"
wget -O "emp-single-end-sequences/sequences.fastq.gz" "https://data.qiime2.org/2017.7/tutorials/moving-pictures/emp-single-end-sequences/sequences.fastq.gz"

# 生成qiime需要的artifact文件(qiime文件格式，将原始数据格式标准化)
qiime tools import \
  --type EMPSingleEndSequences \
  --input-path emp-single-end-sequences \
  --output-path emp-single-end-sequences.qza

输出显示：

$head sample-metadata.tsv
#SampleID   BarcodeSequence LinkerPrimerSequence    BodySite    Year    Month   Day Subject ReportedAntibioticUsage DaysSinceExperimentStarDescription
L1S8    AGCTGACTAGTC    GTGCCAGCMGCCGCGGTAA gut 2008    10  28  subject-1   Yes 0   subject-1.gut.2008-10-28
L1S57   ACACACTATGGC    GTGCCAGCMGCCGCGGTAA gut 2009    1   20  subject-1   No  84  subject-1.gut.2009-1-20
L1S76   ACTACGTGTGGT    GTGCCAGCMGCCGCGGTAA gut 2009    2   17  subject-1   No  112 subject-1.gut.2009-2-17

total 28M
-rw-rw-r-- 1 toucan toucan 3.7M Jul 22  2017 barcodes.fastq.gz
-rw-rw-r-- 1 toucan toucan  25M Jul 22  2017 sequences.fastq.gz

# fastq序列文件
 1 @HWI-EAS440_0386:1:23:17547:1423#0/1
      2 TACGNAGGATCCGAGCGTTATCCGGATTTATTGGGTTTAAAGGGAGCGTAGATGGATGTTTAAGTCAGTTGTGAAAGTTTGCGGCTCAACCGTAAAATTGCAGTTGATACTGGATATCTTGAGTGCAGTTGAGGCAGGGGGGGA
      3 +
      4 IIIE)EEEEEEEEGFIIGIIIHIHHGIIIGIIHHHGIIHGHEGDGIFIGEHGIHHGHHGHHGGHEEGHEGGEHEBBHBBEEDCEDDD>B?BE@@B>@@@@@CB@ABA@@?@@=>?08;3=;==8:5;@6?##############
      5 @HWI-EAS440_0386:1:23:14818:1533#0/1
      6 CCCCNCAGCGGCAAAAATTAAAATTTTTACCGCTTCGGCGTTATAGCCTCACACTCAATCTTTTATCACGAAGTCATGATTGAATCGCGAGTGGTCGGCAGATTGCGATAAACGGGCACATTAAATTTAAACTGATGATTCCAC
      7 +
      8 64<2$24;1)/:*B<?BBDDBBD<>BDD####################################################################################################################

# 数据标准化为qiime2的输入数据
# EMPSingleEndSequences——单端测序、EMPPairedEndSequences——双端测序
# 输入文件夹路径、输出文件
# qza为二进制文件，不能直接打开

qiime tools import --type EMPSingleEndSequences --input-path emp-single-end-sequences  --output-path emp-single-end-sequences.qza

可视化qza文件网站：https://view.qiime2.org/
可视化后显示，

• Peek 记录了 文件的类型
• 流程图

name:"emp-single-end-sequences.qza"
uuid:"207517a2-5d10-43dc-93c3-74a176fcfb6c"
type:"EMPSingleEndSequences"
format:"EMPSingleEndDirFmt"

2. 拆分样品

# 按barcode拆分样品 Demultiplexing sequences
qiime demux emp-single \
  --i-seqs emp-single-end-sequences.qza \
  --m-barcodes-file sample-metadata.tsv \
  --m-barcodes-category BarcodeSequence \
  --o-per-sample-sequences demux.qza

# 结果统计
qiime demux summarize \
  --i-data demux.qza \
  --o-visualization demux.qzv

# 查看结果 (依赖XShell+XManager或其它ssh终端和图形界面软件)
qiime tools view demux.qzv

结果显示：

3. 序列质控和生成OTU表

# 单端序列去噪, 去除左端0bp(--p-trim-left用于切除边缘低质量区)，序列切成120bp长；生成代表序列和OTU表；并重命名用于下游分析
# denoise-single——单端模式
# --i-demultiplexed-seqs 输入序列
# --p-trim-left 左边切除长度为0，等于不切除
# --p-trunc-len 长度过滤最小值
#  --o-representative-sequences 代表序列输出文件路径
#   --o-table 特征标文件路径
# -o-denoising-stats 噪声统计

qiime dada2 denoise-single \
  --i-demultiplexed-seqs 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

mv rep-seqs-dada2.qza rep-seqs.qza
mv table-dada2.qza table.qza

统计文件可视化

qiime metadata tabulate \
  --m-input-file stats-dada2.qza \
  --o-visualization stats-dada2.qzv

统一命名

mv rep-seqs-dada2.qza rep-seqs.qza
mv table-dada2.qza table.qza

4. Feature表统计、代表序列统计

qiime feature-table summarize \
  --i-table table.qza \
  --o-visualization table.qzv \
  --m-sample-metadata-file sample-metadata.tsv
qiime feature-table tabulate-seqs \
  --i-data rep-seqs.qza \
  --o-visualization rep-seqs.qzv

qiime tools view table.qzv
qiime tools view rep-seqs.qzv

table

ref seq

5. 建树：用于多样性分析

# 多序列比对
qiime alignment mafft \
  --i-sequences rep-seqs.qza \
  --o-alignment aligned-rep-seqs.qza
# 移除高变区
qiime alignment mask \
  --i-alignment aligned-rep-seqs.qza \
  --o-masked-alignment masked-aligned-rep-seqs.qza
# 建树
qiime phylogeny fasttree \
  --i-alignment masked-aligned-rep-seqs.qza \
  --o-tree unrooted-tree.qza
# 无根树转换为有根树
qiime phylogeny midpoint-root \
  --i-tree unrooted-tree.qza \
  --o-rooted-tree rooted-tree.qza

6. Aplha多样性

# 指定重抽样的条数，使数据统一标准化，去除过低或过高的样品。标准化采用重抽样至序列一致。 --p-sampling-depth
qiime diversity core-metrics-phylogenetic \
  --i-phylogeny rooted-tree.qza \
  --i-table table.qza \
  --p-sampling-depth 1109 \
  --m-metadata-file sample-metadata.tsv \
  --output-dir core-metrics-results

# 输出结果包括多种多样性结果，文件列表和解释如下：
# beta多样性bray_curtis距离矩阵 bray_curtis_distance_matrix.qza 
# alpha多样性evenness(均匀度，考虑物种和丰度)指数 evenness_vector.qza
# alpha多样性faith_pd(考虑物种间进化关系)指数 faith_pd_vector.qza
# beta多样性jaccard距离矩阵 jaccard_distance_matrix.qza
# 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
# beta多样性unweighted_unifrac距离矩阵，不考虑丰度 unweighted_unifrac_distance_matrix.qza
# beta多样性unweighted_unifrac距离矩阵，考虑丰度 weighted_unifrac_distance_matrix.qza

# 统计faith_pd算法Alpha多样性组间差异是否显著，输入多样性值、实验设计，输出统计结果
qiime diversity alpha-group-significance \
  --i-alpha-diversity core-metrics-results/faith_pd_vector.qza \
  --m-metadata-file sample-metadata.tsv \
  --o-visualization core-metrics-results/faith-pd-group-significance.qzv

# 统计evenness组间差异是否显著
qiime diversity alpha-group-significance \
  --i-alpha-diversity core-metrics-results/evenness_vector.qza \
  --m-metadata-file sample-metadata.tsv \
  --o-visualization core-metrics-results/evenness-group-significance.qzv

# 网页展示结果，只要是qzv的文件，均可用qiime tools view查看或在线https://view.qiime2.org/查看，以后不再赘述
qiime tools view core-metrics-results/faith-pd-group-significance.qzv
qiime tools view core-metrics-results/evenness-group-significance.qzv

faith-pd-group-significance.qzv结果

evenness-group-significance.qzv结果

7.Beta多样性

# 按BodySite分组，统计unweighted_unifrace距离的组间是否有显著差异
qiime diversity beta-group-significance \
  --i-distance-matrix core-metrics-results/unweighted_unifrac_distance_matrix.qza \
  --m-metadata-file sample-metadata.tsv \
  --m-metadata-column BodySite \
  --o-visualization core-metrics-results/unweighted-unifrac-body-site-significance.qzv \
  --p-pairwise

# 按Subject分组，统计unweighted_unifrace距离的组间是否有显著差异
qiime diversity beta-group-significance \
  --i-distance-matrix core-metrics-results/unweighted_unifrac_distance_matrix.qza \
  --m-metadata-file sample-metadata.tsv \
  --m-metadata-column Subject \
  --o-visualization core-metrics-results/unweighted-unifrac-subject-group-significance.qzv \
  --p-pairwise

# 可视化三维展示unweighted-unifrac的主坐标轴分析
qiime emperor plot \
  --i-pcoa core-metrics-results/unweighted_unifrac_pcoa_results.qza \
  --m-metadata-file sample-metadata.tsv \
  --p-custom-axis DaysSinceExperimentStart \
  --o-visualization core-metrics-results/unweighted-unifrac-emperor.qzv

# 可视化三维展示unweighted_unifrac的主坐标轴分析
qiime emperor plot \
  --i-pcoa core-metrics-results/unweighted_unifrac_pcoa_results.qza \
  --m-metadata-file sample-metadata.tsv \
  --p-custom-axes DaysSinceExperimentStart \
  --o-visualization core-metrics-results/unweighted-unifrac-emperor-DaysSinceExperimentStart.qzv

# 可视化三维展示bray-curtis的主坐标轴分析
qiime emperor plot \
  --i-pcoa core-metrics-results/bray_curtis_pcoa_results.qza \
  --m-metadata-file sample-metadata.tsv \
  --p-custom-axes DaysSinceExperimentStart \
  --o-visualization core-metrics-results/bray-curtis-emperor-DaysSinceExperimentStart.qzv

# 网页展示结果，或下载在线查看
qiime tools view core-metrics-results/unweighted-unifrac-emperor-DaysSinceExperimentStart.qzv
qiime tools view core-metrics-results/bray-curtis-emperor-DaysSinceExperimentStart.qzv

unweighted-unifrac

bray-curtis

8. Alpha rarefaction plotting

qiime diversity alpha-rarefaction \
  --i-table table.qza \
  --i-phylogeny rooted-tree.qza \
  --p-max-depth 4000 \
  --m-metadata-file sample-metadata.tsv \
  --o-visualization alpha-rarefaction.qzv

qiime tools view alpha-rarefaction.qzv

9. 物种分类

# 下载物种注释
wget -O "gg-13-8-99-515-806-nb-classifier.qza" "https://data.qiime2.org/2018.4/common/gg-13-8-99-515-806-nb-classifier.qza"

# 物种分类
qiime feature-classifier classify-sklearn \
  --i-classifier gg-13-8-99-515-806-nb-classifier.qza \
  --i-reads rep-seqs.qza \
  --o-classification taxonomy.qza

# 物种结果转换表格，可用于查看
qiime metadata tabulate \
  --m-input-file taxonomy.qza \
  --o-visualization taxonomy.qzv


# 展示taxonomy.qzv结果如下：
qiime tools view taxonomy.qzv
#Feature ID Taxonomy
#d12759fe8dda1d65fe9077cc1ca9cf28   k__Bacteria; p__Bacteroidetes; c__Flavobacteriia; o__Flavobacteriales; f__[Weeksellaceae]; g__Chryseobacterium; s__
#5ada68b9a081358e1a7d5f1d351e656a   k__Bacteria; p__Fusobacteria; c__Fusobacteriia; o__Fusobacteriales; f__Leptotrichiaceae; g__Leptotrichia; s__
#d9095748835ade1b8914c5f57b6acbcf   k__Bacteria; p__Proteobacteria; c__Gammaproteobacteria; o__Aeromonadales; f__Aeromonadaceae; g__Oceanisphaera; s__

# 物种分类柱状图
qiime taxa barplot \
  --i-table table.qza \
  --i-taxonomy taxonomy.qza \
  --m-metadata-file sample-metadata.tsv \
  --o-visualization taxa-bar-plots.qzv

qiime tools view taxa-bar-plots.qzv

10. Differential abundance testing with ANCOM——差异分析

# 只保留肠道样本
qiime feature-table filter-samples \
  --i-table table.qza \
  --m-metadata-file sample-metadata.tsv \
  --p-where "BodySite='gut'" \
  --o-filtered-table gut-table.qza

# # OTU表添加假count，因为ANCOM不允许有零
qiime composition add-pseudocount \
  --i-table gut-table.qza \
  --o-composition-table comp-gut-table.qza

# 采用ancon，按Subject分组进行差异统计
qiime composition ancom \
  --i-table comp-gut-table.qza \
  --m-metadata-file sample-metadata.tsv \
  --m-metadata-column Subject \
  --o-visualization ancom-Subject.qzv

# 查看结果
qiime tools view ancom-Subject.qzv

差异分类学级别分析：以按门水平合并再统计差异

# 按属水平进行合并，统计各门的总reads
qiime taxa collapse \
  --i-table gut-table.qza \
  --i-taxonomy taxonomy.qza \
  --p-level 6 \
  --o-collapsed-table gut-table-l6.qza

# 去除0
qiime composition add-pseudocount \
  --i-table gut-table-l6.qza \
  --o-composition-table comp-gut-table-l6.qza

# # 在属水平按取项目分类部分分析
qiime composition ancom \
  --i-table comp-gut-table-l6.qza \
  --m-metadata-file sample-metadata.tsv \
  --m-metadata-column Subject \
  --o-visualization l6-ancom-Subject.qzv

qiime tools view l6-ancom-Subject.qzv

图片解析有待补充

参考文章：
https://forum.qiime2.org/t/qiime2-chinese-manual/838