文献信息：
标题：Microbial abundance, activity and population genomic profiling with mOTUs2
中文：mOTUs2分析微生物丰度、活力和群落基因组
杂志：Nature Communication
单位：德国海德堡欧洲分子生物学实验室

摘要：

宏基因组测序极大地提高了我们描述环境和宿主相关微生物群落组成的能力。然而，大多数方法依赖参考基因组，这是目前无法为很大一部分微生物物种，引入估计偏差。我们提出了一种基于通用(即参考无关)、系统发育标记基因(MG)为基础的操作分类单元(mOTUs)的更新和功能扩展工具，能够对>7700种微生物进行分析。基于mOTUs的相对丰度估算方法比其他方法更准确，因为有超过30%的相对丰度无法在此分类分辨率下被量化。作为一个新的特征，我们发现mOTUs是基于必需的管家基因，可以很好地量化群落成员的基础转录活性。此外，使用mOTUs估计的单核苷酸变异谱反映了全基因组的变异，这允许比较微生物菌株种群(例如，跨不同人体部位)。

软件地址：https://motu-tool.org/
Github地址：https://github.com/motu-tool/mOTUs_v2

基于通用标记基因的系统发生宏组学分析

2019年更新到2.5版本

工具比较：多样性指数

工具比较：宏基因宏转录相关性

方法比较：SNV分析

测试

github: https://github.com/motu-tool/mOTUs

下载

conda create -n motu-env
conda activate motu-env
conda install -c bioconda python=3.8 bwa samtools
# Download and install mOTUs
pip install motu-profiler
# Download the mOTUs database
motus downloadDB

测试

motus profile --test

运行

# paired end
source /public/home/zzumgg03/huty/softwares/miniconda3/etc/profile.d/conda.sh
conda activate motu-env
mkdir 05_motu/$infile/

for i in `ls clean_merge/$infile/`; do
    motus profile \
    -f clean_merge/$infile/$i/${i}_1.fastq \
    -r clean_merge/$infile/$i/${i}_2.fastq \
    -t 30 > 05_motu/$infile/${i}_motu.txt
done

# motu合并
motus merge \
-i taxonomy_profile_1.txt,taxonomy_profile_2.txt \
> all_sample_profiles.txt

参数：
-f reads in forward orientation, fastq(.gz)-formatted
-r reads in reverse orientation, fastq(.gz)-formatted
-s unpaired reads, fastq(.gz)-formatted

过程

2022-06-25 15:19:23 Map reads to the marker gene database
   Reads are aligned (by BWA) to marker gene sequences in the reference database
   Reads are filtered for:
    - similarity to the reference gene (>97%), and
    - length of the alignment (at least 75 nucleotides, -l option)
   
   Number of detected lanes: 1

   2022-06-25 15:19:23 Run bwa on lane 1
     map forward reads: 3837.77 s
     map reverse reads: 4520.66 s
     sort alignments: 0.04 s
   Total number of reads: 571160793
   Number of reads after filtering: 11351 (0.00 percent)
   
   Total time for mapping reads: 8358.90 s
   
2022-06-25 17:38:42 Calculate marker gene cluster (MGC) abundance
   Abundance of MGCs are quantified by the number of inserts aligning to their
   member genes
   Inserts can either map to one MGC ('Unique mappers'), or map to many
   MGCs from different species ('Multiple mappers')

   Total number of inserts: 10782
     - Unique mappers: 9701
     - Multiple mappers: 1069
     - Ignored multiple mapper without unique hit: 12
   
   Total time to calculate the MGCs: 1401.78 s
   
2022-06-25 18:02:03 Generate mOTU profile
   Each mOTU is composed of 6 to 10 MGCs
   The final mOTU insert count is obtained as the median of its MGC read counts

   At least 3 (-g) MGCs need to be detected to consider a mOTU as being present

   Create taxonomy profile
     Number of ref-mOTUs:  31
     Number of meta-mOTUs: 0
     Number of ext-mOTUs:  5
   
2022-06-25 18:02:05 Finished computation
2022-06-25 18:02:05 Total time: 9762.72 s

结果

一个样本一个文件

所有样本行数都是33574

# git tag version 3.0.2 |  motus version 3.0.2 | map_tax 3.0.2 | gene database: nr3.0.2 | calc_mgc 3.0.2 -y insert.scaled_counts -l 75 | calc_motu 3.0.2 -k mOTU -C no_CAMI -g 3 | taxonomy: ref_mOTU_3.0.2 meta_mOTU_3.0.2
# call: python /public/home/zzumgg03/huty/softwares/miniconda3/envs/motu-env/bin/motus profile -f clean_merge/sample_1/A10/A10_1.fastq -r clean_merge/sample_1/A10/A10_2.fastq -t 30
#consensus_taxonomy     unnamed sample
Leptospira alexanderi [ref_mOTU_v3_00001]       0.0000000000
Leptospira weilii [ref_mOTU_v3_00002]   0.0000000000
Chryseobacterium sp. [ref_mOTU_v3_00004]        0.0000000000
Chryseobacterium gallinarum [ref_mOTU_v3_00005] 0.0000000000
Chryseobacterium indologenes [ref_mOTU_v3_00006]        0.0000000000
Chryseobacterium artocarpi/ureilyticum [ref_mOTU_v3_00007]      0.0000000000
Chryseobacterium jejuense [ref_mOTU_v3_00008]   0.0000000000

结果合并

# motu合并
for i in `ls ./ | grep "sample_*"`; do
    for j in `ls $i`; do
        echo -e "$i/$j" >> motu_samples.list
    done
done
all_samples=`cat motu_samples.list | sed ":a;N;s/\n/,/g;ta"`

motus merge \
-i $all_samples \
> all_sample_profiles.txt

过程

2022-06-27 10:22:38 Merge taxonomic profiles
   Merge multiple mOTU profiles into a taxonomy table, in which rows are mOTUs and
   columns are samples
   
   Number of detected files: 150
   
2022-06-27 10:22:53 Finished computation
2022-06-27 10:22:53 Total time: 14.53 s