今天发现了一个扩增子分析的流程Comeau et al., 2017 Microbiome Helper: a Custom and Streamlined Workflow for Microbiome Research. mSystems. Microbiome Helper相当于一个资源库，整和了很多软件，脚本和教程。主要有以下特点：
1. 一系列的可用于处理和自动化操作各种微生物组和宏基因组的脚本构成的生物信息工具。
2. 可用于处理16S/18S rRNA 和宏基因组数据的工作流程或标准工作程序(SOPs)。
3. 测试数据，范例的输出以及各种不同微生物组分析的问题的专题教程。
4. 一个几乎无需配置的能被用作运行分析流程的Virtual Box镜像。
一、软件要求：
Both pipelines
FastQC (optional): http://www.bioinformatics.babraham.ac.uk/projects/fastqc/
PEAR: http://sco.h-its.org/exelixis/web/software/pear/doc.html
GNU Parallel (>= v20170322): https://www.gnu.org/software/parallel/
Metagenomics
Bowtie2: http://bowtie-bio.sourceforge.net/bowtie2/index.shtml
DIAMOND (> v.0.7.0): http://ab.inf.uni-tuebingen.de/software/diamond/
Human pre-indexed database: ftp://ftp.ccb.jhu.edu/pub/data/bowtie2_indexes/hg19.zip
HUMAnN2: http://huttenhower.sph.harvard.edu/humann2
kneadData: https://bitbucket.org/biobakery/kneaddata/wiki/Home
MetaPhlAn2: https://bitbucket.org/biobakery/metaphlan2
Trimmomatic: http://www.usadellab.org/cms/?page=trimmomatic
16S
FASTX toolkit (v0.0.14): http://hannonlab.cshl.edu/fastx_toolkit/download.html
BBMap: http://sourceforge.net/projects/bbmap
VSEARCH: https://github.com/torognes/vsearch
QIIME: http://qiime.org
SortMeRNA: http://bioinfo.lifl.fr/RNA/sortmerna/
SUMACLUST: http://metabarcoding.org/sumatra
PICRUSt: http://picrust.github.io/picrust/
Visualization
STAMP: http://kiwi.cs.dal.ca/Software/STAMP
Databases (dropbox download links)
16S chimera checking: RDP_trainset16_022016.fa (20 MB). This DB was originally from the Ribosome Database Project (RDP) and was parsed to include only bacteria.
18S chimera checking: Eukaryota_SILVA_123_SSURef_Nr99_tax_silva_U-replaced.fa (117 MB). This DB was taken from SILVA and was parsed to include only eukaryotes and all Us were converted to Ts.
ITS2 chimera checking: UNITE_uchime_ITS2only_01.01.2016.fasta (8.6 MB) taken from UNITE database.
Eukaryotic OTU-picking files (from the PR2 database):
gb203_pr2_all_10_28_99p_tax_Xs-fixed_poly-fixed.txt (9 MB)
gb203_pr2_all_10_28_99p_clean.fasta (98 MB)
90_Silva_111_rep_set_euk_aligned.filter.fasta (95 MB)
ITS2 OTU-picking files (taken from UNITE database):
UNITE_sh_refs_qiime_ver7_dynamic_20.11.2016.fasta (16.9 MB).
UNITE_sh_refs_qiime_ver7_dynamic_20.11.2016.goodASCII.txt (4.2 MB) - we converted all non-ASCII characters to ASCII format.
To use "run_contaminant_filter.pl" you will need to download the appropriate Bowtie2 index.
To use "run_pre_humann.pl" you will need to use a database of KEGG orthologs. You may also want to download the raw FASTA.
Perl modules
File::Basename Getopt::Long List::Util Parallel::ForkManager Pod::Usage
二、Microbiome Helper Virtual Box虚拟机使用
1. 硬件要求：64位系统，至少4G内存， 扩增子版本虚拟机镜像文件8G（输入镜像文件20G），完整的 Microbiome Helper镜像文件20G(输入镜像文件20G)。
2. 安装
① 下载安装适合你自己电脑的VirtualBox https://www.virtualbox.org/wiki/Downloads。
② 下载你需要的Microbiome Helper的镜像版本，Microbiome Helper Vbox Amplicon-only (v0.3) (8 GB - OVA file)，Microbiome Helper Vbox (v2.2.1) (20 GB - OVA file)。
③ 将镜像文件安装到VirtualBox中，方法可百度。
④如果你对电脑内存是8G或更大，推荐设置虚拟机内存为4096MB，CPU核数≥2。
3. 其他
默认的用户名和密码为mh_user
今天就写到这里，之前在学习DADA2的使用，还没有很好的进展，下次来记录一下DADA2的使用过程。
