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GGMP

Population-based survey linking gut microbiome to economic development and metabolic syndrome

Introduction

Analysis pipeline for: Population-based survey linking gut microbiome to economic development and metabolic syndrome

Author

     Author:     Hui-Min Zheng, Pan Li, Xian Wang and Yan He    
Last update:     2017-04-18   
      Email:     328093402@qq.com   

Index


1 Environment
1.1 System
1.1.1 System Platform
1.1.2 Hardware
1.2 R
1.2.1 R version
1.2.2 R libraries
1.3 MaAsLin
1.4 Qiime
1.4.1 Qiime version
1.4.2 System information
1.4.3 QIIME default reference information
1.4.4 QIIME config values
1.5 BBMap

2 Data
2.1 original sequences
2.2 metadata

3 Scripts
3.1 Perl Scripts
3.2 R Scripts

4 Supplementary files
4.1 metadata_category.txt
4.2 taxa.list

5 Direction for use
5.1 Configuring the system environment files and variables
5.2 Location of the files
5.3 modify Relate_metadata_with_microbiota.pl
5.4 Run pipeline

6 How to get the data?
6.1 How to get the original sequences?
6.2 How to get the metadata?

1 Environment

1.1 System


1.1.1 System Platform


Platform:      Linux2 
 Version:      Linux version 2.6.32-573.8.1.el6.x86_64 (mockbuild@c6b8.bsys.dev.centos.org) (gcc version 4.4.7 20120313 (Red Hat 4.4.7-16)(GCC))
      OS:      CentOS release 6.7 (Final)

1.1.2 Hardware


     Cpu(s):      >10
     thread:      >10
        RAM:      >10G
  Hard disk:      >2T

1.3 R


1.3.1 R version


    Version:      R version 3.2.5(Linux)(2016-04-14)(www.r-project.org)
  Copyright:      (C) 2016 The R Foundation for Statistical Computing

1.3.2 R libraries


  ggplot2
  psych
  reshape
  agricolae
  gam
  gamlss
  gbm
  glmnet
  inlinedocs
  logging
  MASS
  nlme (version 3.1-127)
  optparse
  outliers
  penalized
  pscl
  robustbase

1.3 MaAsLin


(https://bitbucket.org/biobakery/maaslin/)
       Package: Maaslin
          Type: Package
         Title: Maaslin
       Version: 0.0.3
       Imports: agricolae, gam, gamlss, gbm, glmnet, inlinedocs, logging, MASS, nlme, optparse, outliers, penalized, pscl, robustbase
          Date: 2014-12-04
        Author: Timothy Tickle<ttickle@hsph.harvard.edu>, Curtis Huttenhower <chuttenh@hsph.harvard.edu>
    Maintainer: Timothy Tickle <ttickle@hsph.harvard.edu>,Ayshwarya
                Subramanian<subraman@broadinstitute.org>,Lauren
                McIver<lauren.j.mciver@gmail.com>,George
                Weingart<george.weingart@gmail.com>
   Description: MaAsLin is a multivariate statistical framework that finds associations between clinical metadata and microbial   community abundance or function. The clinical metadata can be of any type continuous (for example age and weight), boolean (sex, stool/biopsy), or discrete/factor (cohort groupings and phenotypes).  MaAsLin is best used in the case when you are associating many metadata with microbial measurements. When this is the case each metadatum can be a diffrent type.  For example, you could include age, weight, sex, cohort and phenotype in the same input file to be analyzed in the same MaAsLin run. The microbial measurements are expected to be normalized before using MaAsLin and so are proportional data ranging from 0 to 1.0
        License: MIT + file LICENSE
VignetteBuilder: knitr
       Suggests: knitr, BiocStyle, BiocGenerics
      biocViews: Statistics, Metagenomics, Bioinformatics, Software
       Packaged: 2015-04-02 00:28:13 UTC; gweingart

1.4 Qiime


1.4.1 Qiime version


  Version:      qiime 1.9.1

1.4.2 System information


           Platform:      Linux2
     Python version:      2.7.10 (default, Dec  4 2015, 15:36:19)  [GCC 4.4.7 20120313 (Red Hat 4.4.7-16)]
  Python executable:      /usr/local/bin/python

1.4.3 QIIME default reference information


For details on what files are used as QIIME's default references, see here:
https://github.com/biocore/qiime-default-reference/releases/tag/0.1.3

          QIIME library version:      1.9.1
           QIIME script version:      1.9.1
qiime-default-reference version:      0.1.3
                  NumPy version:      1.11.0
                  SciPy version:      0.17.1
                 pandas version:      0.17.1
             matplotlib version:      1.4.3
            biom-format version:      2.1.5
                   qcli version:      0.1.1
                   pyqi version:      0.3.2
             scikit-bio version:      0.2.3
                 PyNAST version:      1.2.2
                Emperor version:      0.9.51
                burrito version:      0.9.1
       burrito-fillings version:      0.1.1
              sortmerna version:      SortMeRNA version 2.0, 29/11/2014
              sumaclust version:      SUMACLUST Version 1.0.00
                  swarm version:      Swarm 1.2.19 [Dec  5 2015 16:48:11]
                          gdata:      Installed.

1.4.4 QIIME config values


For definitions of these settings and to learn how to configure QIIME, see here:
http://qiime.org/install/qiime_config.html
http://qiime.org/tutorials/parallel_qiime.html

QIIME config values

For definitions of these settings and to learn how to configure QIIME, see here:
http://qiime.org/install/qiime_config.html
http://qiime.org/tutorials/parallel_qiime.html

                      blastmat_dir:      None
       pick_otus_reference_seqs_fp:      /usr/local/lib/python2.7/sitepackages/qiime_default_reference/gg_13_8_otus/rep_set/97_otus.fasta
                     python_exe_fp:      python
                          sc_queue:      all.q
       topiaryexplorer_project_dir:      None
      pynast_template_alignment_fp:      /usr/local/data/core_set_aligned.fasta.imputed
                   cluster_jobs_fp:      None
 pynast_template_alignment_blastdb:      None
 assign_taxonomy_reference_seqs_fp:      /usr/local/lib/python2.7/sitepackages/qiime_default_reference/gg_13_8_otus/rep_set/97_otus.fasta
                      torque_queue:      friendlyq
               qiime_test_data_dir:      None
    template_alignment_lanemask_fp:      /usr/local/data/lanemask_in_1s_and_0s.txt
                     jobs_to_start:      1
                        slurm_time:      None
                 cloud_environment:      False
                 qiime_scripts_dir:      /usr/local/bin
             denoiser_min_per_core:      50
                       working_dir:      None
 assign_taxonomy_id_to_taxonomy_fp:      /usr/local/lib/python2.7/sitepackages/qiime_default_reference/gg_13_8_otus/taxonomy/97_otu_taxonomy.txt
                          temp_dir:      /tmp/
                      slurm_memory:      None
                       slurm_queue:      None
                       blastall_fp:      blastall
                  seconds_to_sleep:      2

1.5 BBMap


BBTools bioinformatics tools, including BBMap.
Author: Brian Bushnell, Jon Rood
Language: Java
Version 36.32

2 Data


2.1 original sequences


 format of sequences :      fastq
   Number of fq files:      36
         fq_filenames:      1.Clean_FCHVJVMBCXX_L1_wHAXPI034525-109_1.fq
                            1.Clean_FCHVJVMBCXX_L1_wHAXPI034525-109_2.fq
                            1.Clean_FCHVJVMBCXX_L2_wHAXPI034525-109_1.fq
                            1.Clean_FCHVJVMBCXX_L2_wHAXPI034525-109_2.fq
                            1.Clean_FCHVTWCBCXX_L1_wHAXPI034526-108_1.fq
                            1.Clean_FCHVTWCBCXX_L1_wHAXPI034526-108_2.fq
                            1.Clean_FCHVTWCBCXX_L2_wHAXPI034526-108_1.fq
                            1.Clean_FCHVTWCBCXX_L2_wHAXPI034526-108_2.fq
                            2.Clean_FCHVJVMBCXX_L1_wHAXPI034525-109_1.fq
                            2.Clean_FCHVJVMBCXX_L1_wHAXPI034525-109_2.fq
                            2.Clean_FCHVJVMBCXX_L2_wHAXPI034525-109_1.fq
                            2.Clean_FCHVJVMBCXX_L2_wHAXPI034525-109_2.fq
                            2.Clean_FCHVTWCBCXX_L1_wHAXPI034526-108_1.fq
                            2.Clean_FCHVTWCBCXX_L1_wHAXPI034526-108_2.fq
                            2.Clean_FCHVTWCBCXX_L2_wHAXPI034526-108_1.fq
                            2.Clean_FCHVTWCBCXX_L2_wHAXPI034526-108_2.fq
                            3.Clean_FCHVJVMBCXX_L1_wHAXPI034525-109_1.fq
                            3.Clean_FCHVJVMBCXX_L1_wHAXPI034525-109_2.fq
                            3.Clean_FCHVJVMBCXX_L2_wHAXPI034525-109_1.fq
                            3.Clean_FCHVJVMBCXX_L2_wHAXPI034525-109_2.fq
                            3.Clean_FCHVTWCBCXX_L1_wHAXPI034526-108_1.fq
                            3.Clean_FCHVTWCBCXX_L1_wHAXPI034526-108_2.fq
                            3.Clean_FCHVTWCBCXX_L2_wHAXPI034526-108_1.fq
                            3.Clean_FCHVTWCBCXX_L2_wHAXPI034526-108_2.fq
                            4.Clean_FCHVJVMBCXX_L1_wHAXPI034525-109_1.fq
                            4.Clean_FCHVJVMBCXX_L1_wHAXPI034525-109_2.fq
                            4.Clean_FCHVJVMBCXX_L2_wHAXPI034525-109_1.fq
                            4.Clean_FCHVJVMBCXX_L2_wHAXPI034525-109_2.fq
                            4.Clean_FCHVTWCBCXX_L1_wHAXPI034526-108_1.fq
                            4.Clean_FCHVTWCBCXX_L1_wHAXPI034526-108_2.fq
                            4.Clean_FCHVTWCBCXX_L2_wHAXPI034526-108_1.fq
                            4.Clean_FCHVTWCBCXX_L2_wHAXPI034526-108_2.fq
                            5.Clean_FCHVJVMBCXX_L1_wHAXPI034525-109_1.fq
                            5.Clean_FCHVJVMBCXX_L1_wHAXPI034525-109_2.fq
                            5.Clean_FCHVJVMBCXX_L2_wHAXPI034525-109_1.fq
                            5.Clean_FCHVJVMBCXX_L2_wHAXPI034525-109_2.fq

2.2 metadata


 Filename:       Additional file 2: Table S1
   Header:       #SampleID	county_level_code	age	gender	Bristol_stool_type	fam_income_year_avg	fam_spend_year_avg
                  MetS	anthrop_waist	anthrop_SBP	anthrop_DBP	biochem_FBG	biochem_TG	biochem_HDL
Row.names:       6896 SampleIDs

3 Scripts


3.1 Perl Scripts


3.1.1 Preprocessing.pl


     Function:      Pipline of preprocessing, this script performs all processing steps through building the OTU table with several pair of fastq file.
  Last updata:      2016-09-18
       Author:      Huimin Zheng

3.1.2 Relate_metadata_with_microbiota.pl


     Function:      Pipline of geting main results of the paper-Population-based survey linking gut microbiome to economic development and metabolic syndrome.
  Last updata:      2017-04-18
       Author:      Huimin Zheng

3.1.3 Illumina_pairend_preprocessing.pl


     Function:      This script performs all processing steps through building the OTU table with one pair of fastq file.
     Location:      Called by the pipeline--Preprocessing.pl
  Last updata:      2016-08-08
       Author:      Yan He

3.1.4 trim_200bp.pl


     Function:      Trim the fastq file to 200bp, this can reduce the computational burden while using enough information to do overlapping
     Location:      Called by the script--Illumina_pairend_preprocessing.pl
  Last updata:      2016-08-08
       Author:      Hua-Fang Sheng

3.1.5 pairend.extract_sequences.pl


     Function:      Do library splitting, as barcodes on both ends is not quite supported by QIIME at the moment (QIIME 1.9.1)
     Location:      Called by the script--Illumina_pairend_preprocessing.pl
  Last updata:      2016-08-08
       Author:      Yan He

3.1.6 merge_adiv_metadata.pl


     Function:      merge the alpha diversity file and metadata file.
     Location:      Called by the pipeline--Relate_metadata_with_microbiota.pl
  Last updata:      2016-08-08
       Author:      Yan He

3.1.7 adonis_dilution_curve.pl


     Function:      Examined the significance using adonis analysis on various subsampling sizes with specific replications at each size.
     Location:      Called by the pipeline--Relate_metadata_with_microbiota.pl
  Last updata:      2017-04-16
       Author:      Huimin Zheng

3.1.8 adonis_all_metadata.pl


     Function:      Do adonis analysis on all variables supplied. 
     Location:      Called by the script--adonis_dilution_curve.pl
  Last updata:      2016-08-08
       Author:      Yan He

3.1.9 adonis_dilution_curve_plot.pl


     Function:      Collect the adonis analysis results of script adonis_dilution_curve.pl and plot.
     Location:      Called by the script--Illumina_pairend_preprocessing.pl
  Last updata:      2017-04-16
       Author:      Huimin Zheng

3.1.10 maaslin_and_cytoscape.otu.pl


     Function:      Do MaAsLin analysis
     Location:      Called by the pipeline--Relate_metadata_with_microbiota.pl
  Last updata:      2016-08-08
       Author:      Yan He

3.1.11 add_taxa_to_map.pl


     Function:      Add relative abundance of taxa in taxa.list to metadata file.
     Location:      Called by the pipeline--Relate_metadata_with_microbiota.pl
  Last updata:      2017-04-16
       Author:      Huimin Zheng

3.1.12 collapsed_metadata.pl


     Function:      Collapse samples in metadata file. Values in the metadata file are collapsed by means of continuous variables and proportion of categorical variables for each group.
     Location:      Called by the pipeline--Relate_metadata_with_microbiota.pl
  Last updata:      2017-04-16
       Author:      Huimin Zheng

3.2 R Scripts


3.2.1 MetS_incidences_between_quartiles.R


     Function:      Calculate MetS incidences between quartiles and plot
     Location:      Called by the pipeline--Relate_metadata_with_microbiota.pl
  Last updata:      2017-04-16
       Author:      Pan Li

4 Supplementary files


4.1 metadata_category.txt


    Supplementary file of maaslin_and_cytoscape.otu.pl

4.2 taxa.list


    Supplementary file of add_taxa_to_map.pl

5 Direction for use


5.1 Configuring the system environment files and variables


   Configuring the system environment files and variables based on the (1) Environment

5.2 Location of the files


  put the scripts, bbmap folder and supplementary files in the same path
  put all fastq files in the same path

5.3 modify Relate_metadata_with_microbiota.pl


line 39:   get the path of 97_otus.fasta, such as /usr/local/lib/python2.7/site-packages/qiime_default_reference/gg_13_8_otus/rep_set/97_otus.fasta
line 51:   get the path of 97_otus.fasta, such as /usr/local/lib/python2.7/site-packages/qiime_default_reference/gg_13_8_otus/rep_set/97_otus.fasta

5.4 Run pipeline


5.4.1 Preprocessing: From raw sequences to BIOM


 perl Preprocessing.pl <fq_dir> <metadata.list> <threads> <output_dir> 
 <fq_dir>: Path to the folder containing all fastq files.
 <metadata.list>: Path to file listing path to metadata file.
 <threads>: Specify number of threads.
 <output_dir>: The output directory.
 # nohup perl Preprocessing.pl <fq_dir> <metadata.list> <threads> <output_dir> > Preprocessing.log 2>&1 &

5.4.2 Relate metadata with microbiota: From BIOM to downstream results


 perl Relate_metadata_with_microbiota.pl <otu_table.biom> <metadata> <output_dir>
 <otu_table.biom>: The input otu table filepath in biom format.
 <metadata>: Path to the metadata file.
 <output_dir>: The output directory.
 #nohup perl Relate_metadata_with_microbiota.pl <otu_table.biom> <metadata> <output_dir> > Preprocessing.log 2>&1 &

6 How to get the data?


6.1 How to get the original sequences?


 The 16S gene sequencing reads of GGMP have been deposited in EBI under accession PRJEB18535.   

6.2 How to get the metadata?


 Please contact Prof. Hong-Wei Zhou(biodegradation@gmail.com) to get a application form for access to the GGMP metadata.        

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