MetaPhlAn4宏基因组数据分析流程学习笔记

参考文献

Blanco-Míguez, A., Beghini, F., Cumbo, F. et al. Extending and improving metagenomic taxonomic profiling with uncharacterized species using MetaPhlAn 4. Nat Biotechnol (2023).

官方网站

MetaPhlAn 4.0

软件安装

推荐使用mamba安装：

mamba create --name metaphlan4
mamba activate metaphlan4
mamba install -c bioconda metaphlan

数据库下载

默认第一次运行软件会自动下载数据库，也可以手动下载：

nohup axel -n 60 http://cmprod1.cibio.unitn.it/biobakery4/metaphlan_databases/bowtie2_indexes/mpa_vOct22_CHOCOPhlAnSGB_202212_bt2.tar &

wget http://cmprod1.cibio.unitn.it/biobakery4/metaphlan_databases/mpa_vOct22_CHOCOPhlAnSGB_202212.tar

下载完成解压后放到默认的目录下即可，默认目录是：~/mambaforge/envs/metaphlan4/lib/python3.10/site-packages/metaphlan/metaphlan_databases.

下载完成后运行下面的代码会自动解压并创建数据库：

metaphlan --install

开始分析

输入文件

支持fasta、fastq、sam和bowtie2out这四种格式文件作为输入文件。

示例数据

• SRS014476-Supragingival_plaque.fasta.gz
• SRS014494-Posterior_fornix.fasta.gz
• SRS014459-Stool.fasta.gz
• SRS014464-Anterior_nares.fasta.gz
• SRS014470-Tongue_dorsum.fasta.gz
• SRS014472-Buccal_mucosa.fasta.gz

运行单个fastq文件

metaphlan --offline --nproc 60 data/SRS014476-Supragingival_plaque.fasta.gz --input_type fasta > output/SRS014476-Supragingival_plaque_profile.txt  

输出文件

• Bowtie2的中间文件：比对结果。

❯ head data/SRS014476-Supragingival_plaque.fasta.gz.bowtie2out.txt                                                                                       ─╯
HWUSI-EAS1568_102539179:1:100:10007:7282/1__1.50        UniRef90_P44049|1__7|SGB9649
HWUSI-EAS1568_102539179:1:100:10008:17064/1__1.53       UniRef90_E0DI50|1__4|SGB17007
HWUSI-EAS1568_102539179:1:100:10012:9508/1__1.85        UniRef90_A0A3S4XT37|1__5|SGB17007
HWUSI-EAS1568_102539179:1:100:10013:7741/1__1.92        UniRef90_UNK19880-CIDAPOPB_00170|1__8|SGB19880
HWUSI-EAS1568_102539179:1:100:10015:15592/1__1.105      SGB6007__DMIBLHOP_01812
HWUSI-EAS1568_102539179:1:100:10025:5272/1__1.159       UniRef90_A0A3S5F533|1__7|SGB17007
HWUSI-EAS1568_102539179:1:100:10035:9129/1__1.208       UniRef90_UNK69135-EJPKGDGA_01179|7__12|SGB69135
HWUSI-EAS1568_102539179:1:100:10036:1783/1__1.212       UniRef90_A0A2A8D7T0|1__5|SGB49305
HWUSI-EAS1568_102539179:1:100:10039:18013/1__1.234      UniRef90_UNK98242-PKBBBOLB_00132|3__13|SGB98242
HWUSI-EAS1568_102539179:1:100:10047:18261/1__1.293      UniRef90_I0UTJ6|1__5|SGB16987

• MetaPhlAn的分类文件:起始也就是每个物种在每个样品中的丰度。

head output/SRS014476-Supragingival_plaque_profile.txt  
#mpa_vOct22_CHOCOPhlAnSGB_202212
#/home/lixiang/mambaforge/envs/metaphlan4/bin/metaphlan --offline --nproc 60 data/SRS014476-Supragingival_plaque.fasta.gz --input_type fasta
#19048 reads processed
#SampleID       Metaphlan_Analysis
#clade_name     NCBI_tax_id     relative_abundance      additional_species
k__Bacteria     2       100.0
k__Bacteria|p__Actinobacteria   2|201174        55.36506
k__Bacteria|p__Firmicutes       2|1239  44.63494
k__Bacteria|p__Actinobacteria|c__Actinomycetia  2|201174|1760   55.36506
k__Bacteria|p__Firmicutes|c__Bacilli    2|1239|91061    44.63494

重新分析样本

如果要重新分析样本的话，直接使用上一步输出的bowtie2结果就好。省去比对步骤，速度更快。

metaphlan --offline --nproc 60 data/SRS014476-Supragingival_plaque.fasta.gz.bowtie2out.txt --input_type bowtie2out > output/SRS014476-Supragingival_plaque_profile.txt

多个样本运行

for i in SRS*.fasta.gz; do metaphlan $i --input_type fasta --nproc 60 --offline > ${i%.fasta.gz}_profile.txt; done

merge_metaphlan_tables.py output/* > merged.abundance.table.txt

数据可视化

分类单元热图

grep -E "s__|SRS" merged_abundance_table.txt \
| grep -v "t__" \
| sed "s/^.*|//g" \
| sed "s/SRS[0-9]*-//g" \
> merged_abundance_table_species.txt