DOC for V1.0.5

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Contents

Introduction

PGCGAP is a pipeline for prokaryotic comparative genomics analysis. It can take the pair-end reads as input. In addition to genome assembly, gene prediction and annotation, it can also get common comparative genomics analysis results such as phylogenetic trees of single-core proteins and core SNPs, pan-genome, whole-genome Average Nucleotide Identity (ANI), orthogroups and orthologs, COG annotations, substitutions (snps) and insertions/deletions (indels) with only one line of commands.

Installation

The software was tested successfully on Linux x64 platform and macOS. However, Windows could not be supported. Because this software relies on a large number of other softwares, so it is recommended to install with Bioconda. The main program and most of other dependencies can be installed with one command as shown below, but the "Gubbins" should be installed separately because of it relys on python3, while PGCGAP relys on python2.

Step1: Install Gubbins

$conda install gubbins

Step2: Install PGCGAP

$conda create -n pgcgap python=2.7 anaconda
$conda activate pgcgap
$conda install pgcgap # By command "whereis pgcgap", users can find the installation path of pgcgap, and then you should add it into your environment variable.
$conda deactivate

Step3: Setup COG database (Users should execute this after first installation of pgcgap)

$pgcgap --setup-COGdb

Required dependencies

Usage

  • Print the help messages: 
      $pgcgap --help


  • General usage: 
      $pgcgap [Fuctions] [options]


  • Setup COG database: (Users should execute this after first installation of pgcgap) 
      $pgcgap --setup-COGdb


  • FUCTIONS:

    • [–All] Perform Assemble, CoreTree, Pan, OrthoF, ANI and COG functions with one command

    • [–Assemble] Assemble reads ( paired-end only ) into contigs, predict genes and annotate them

    • [–CoreTree] Construct single-core proteins tree and SNPs tree of single core genes

    • [–Pan] Run "roary" pan genome pipeline with gff3 files

    • [–OrthoF] Identify orthologous protein sequence families with "OrthoFinder"

    • [–ANI] Compute whole-genome Average Nucleotide Identity ( ANI )

    • [–COG] Run COG annotation for each strain (*.faa), and generate a table containing the relative abundance of each flag for all strains

    • [–VAR] Rapid haploid variant calling and core genome alignment with "Snippy"

  • Global Options:

    • [–strain_num (INT)] [Required by "–All", "–CoreTree" and "–VAR"] The total number of strains used for analysis, including reference genomes

    • [–ReadsPath (PATH)] [Required by "–All", "–Assemble" and "–VAR"] Reads of all strains as file paths ( Default ./Reads )

    • [–AAsPath (PATH)] [Required by "–CoreTree" and "–COG"] Amino acids of all strains as fasta file paths, ( Default "./Results/Annotations/AAs" )

    • [–reads1 (STRING)] [Required by "–All", "–Assemble" and "–VAR"] The suffix name of reads 1 ( for example: if the name of reads 1 is "YBT-1520_L1_I050.R1.clean.fastq.gz", "YBT-1520" is the strain same, so the suffix name should be ".R1.clean.fastq.gz")

    • [–reads2 (STRING)] [Required by "–All", "–Assemble" and "–VAR"] The suffix name of reads 2( for example: if the name of reads 2 is "YBT-1520_2.fq", the suffix name should be "_2.fq" )

    • [–codon (INT)] [Required by "–All", "–Assemble", "–CoreTree" and "–Pan"] Translation table ( Default 11 )

      • 1 Universal code
      • 2 Vertebrate mitochondrial code
      • 3 Yeast mitochondrial code
      • 4 Mold, Protozoan, and Coelenterate Mitochondrial code and Mycoplasma/Spiroplasma code
      • 5 Invertebrate mitochondrial
      • 6 Ciliate, Dasycladacean and Hexamita nuclear code
      • 9 Echinoderm and Flatworm mitochondrial code
      • 10 Euplotid nuclear code
      • 11 Bacterial, archaeal and plant plastid code ( Default )
      • 12 Alternative yeast nuclear code
      • 13 Ascidian mitochondrial code
      • 14 Alternative flatworm mitochondrial code
      • 15 Blepharisma nuclear code
      • 16 Chlorophycean mitochondrial code
      • 21 Trematode mitochondrial code
      • 22 Scenedesmus obliquus mitochondrial code
      • 23 Thraustochytrium mitochondrial code

    • [–suffix_len (INT)] [Required by "–All", "–Assemble" and "–VAR"] (Strongly recommended) The suffix length of the reads, that is the length of your reads name minus the length of your strain name. For example the –suffix_len of "YBT-1520_L1_I050.R1.clean.fastq.gz" is 26 ( "YBT-1520" is the strain name ) ( Default 0 )

    • [–logs (STRING)] Name of the log file ( Default Logs.txt )

    • [–threads (INT)] Number of threads to be used ( Default 4 )

  • Local Options:

    • –Assemble

      • [–kmmer (INT)] [Required] k-mer size for genome assembly ( Default 81 )

      • [–genus (STRING)] Genus name of your strain ( Default "NA" )

      • [–species (STRING)] Species name of your strain ( Default "NA")

    • –CoreTree

      • [–CDsPath (PATH)] [Required] CDs of all strains as fasta file paths, ( Default "./Results/Annotations/CDs" )

      • [-c (FLOAT)] Sequence identity threshold, ( Default 0.5)

      • [-n (INT)] Word_length, see user's guide of CD-HIT for choosing it ( Default 2 )

      • [-G (INT)] Use global (set to 1) or local (set to 0) sequence identity, ( Default 0 )

      • [-t (INT)] Tolerance for redundance ( Default 0 )

      • [-aL (FLOAT)] Alignment coverage for the longer sequence. If set to 0.9, the alignment must covers 90% of the sequence ( Default 0.9 )

      • [-aS (FLOAT)] Alignment coverage for the shorter sequence. If set to 0.9, the alignment must covers 90% of the sequence ( Default 0.9 )

      • [-g (INT)] If set to 0, a sequence is clustered to the first cluster that meet the threshold (fast cluster). If set to 1, the program will cluster it into the most similar cluster that meet the threshold (accurate but slow mode, Default 1)

      • [-d (INT)] length of description in .clstr file. if set to 0, it takes the fasta defline and stops at first space ( Default 0 )

    • –Pan

      • [–GffPath (PATH)] [Required] Gff files of all strains as paths ( Default "./Results/Annotations/GFF" )

    • –orthoF

      • [–Sprogram (STRING)] Sequence search program, Options: blast, mmseqs, blast_gz, diamond ( Default blast)

    • –ANI

      • [–queryL (FILE)] [Required] The file containing paths to query genomes, one per line ( Default scaf.list )

      • [–refL (FILE)] [Required] The file containing paths to reference genomes, one per line. ( Default scaf.list )

      • [–ANIO (FILE)] The name of output file ( Default "Results/ANI/ANIs" )

      • [–Scaf_suffix (STRING)] The suffix of scaffolds or genomes ( Default "-8.fa" )

    • –VAR

      • [–refgbk (FILE)] [Required] The full path and name of reference genome in GENBANK format ( recommended ), fasta format is also OK. For example: "/mnt/g/test/ref.gbk"

      • [–qualtype (STRING)] [Required] Type of quality values (solexa (CASAVA < 1.3), illumina (CASAVA 1.3 to 1.7), sanger (which is CASAVA >= 1.8)). ( Default sanger )

      • [–qual (INT)] Threshold for trimming based on average quality in a window. ( Default 20 )

      • [–length (INT)] Threshold to keep a read based on length after trimming. ( Default 20 )

      • [–mincov (INT)] The minimum number of reads covering a site to be considered ( Default 10 )

      • [–minfrac (FLOAT)] The minimum proportion of those reads which must differ from the reference ( Default 0.9 )

      • [–minqual (INT)] The minimum VCF variant call "quality" ( Default 100 )

      • [–ram (INT)] Try and keep RAM under this many GB ( Default 8 )

      • [–tree_builder (STRING)] Application to use for tree building [raxml|fasttree|hybrid] ( Default fasttree)

      • [–iterations (INT)] Maximum No. of iterations for gubbins ( Default 5 )

  • Paths of external programs

    Not needed if they were in the environment variables path. Users can check with the "–check-external-programs" option for the essential programs.

    • [–abyss-bin (PATH)] Path to abyss binary file. Default tries if abyss is in PATH;

    • [–prodigal-bin (PATH)] Path to prodigal binary file. Default tries if prodigal is in PATH;

    • [–prokka-bin (PATH)] Path to prokka binary file. Default tries if prokka is in PATH;

    • [–cd-hit-bin (PATH)] Path to cd-hit binary file. Default tries if cd-hit is in PATH;

    • [–mafft-bin (PATH)] Path to mafft binary file. Default tries if mafft is in PATH;

    • [–fasttree-bin (PATH)] Path to the fasttree binary file. Default tries if fasttree is in PATH;

    • [–pal2nal-bin (PATH)] Path to the pal2nal.pl binary file. Default tries if pal2nal.pl is in PATH;

    • [–snp-sites-bin (PATH)] Path to the snp-sites binary file. Default tries if snp-sites is in PATH;

    • [–roary-bin (PATH)] Path to the roary binary file. Default tries if roary is in PATH;

    • [–orthofinder-bin (PATH)] Path to the orthofinder binary file. Default tries if orthofinder is in PATH;

    • [–fastANI-bin (PATH)] Path to the fastANI binary file. Default tries if fastANI is in PATH;

    • [–gubbins-bin (PATH)] Path to the run_gubbins.py binary file. Default tries if run_gubbins.py is in PATH;

    • [–snippy-bin (PATH)] Path to the snippy binary file. Default tries if snippy is in PATH;

    • [–sickle-bin (PATH)] Path to the sickle-trim binary file. Default tries if sickle is in PATH.

  • Setup COG database

    • [–setup-COGdb] Users should execute this after first installation of pgcgap.
  • Check the required external programs (It is strongly recommended that this step be performed after the installation of pcgp): 
      $pgcgap --check-external-programs


  • EXAMPLES:

    • Example 1: Perform all functions, take the bacillus thuringiensis as an example, total 4 strains for analysis.

      Notice: For the sake of flexibility, The "VAR" function needs to be added additionally.

      $pgcgap --All --ReadsPath <PATH> --reads1 .R1.clean.fastq.gz --reads2 .R2.clean.fastq.gz --suffix_len <INT> --kmmer 81 --genus bacillus --species thuringiensis --codon 11 --strain_num 4 --threads 4 --VAR --refgbk <FILE> --qualtype <STRING>

    • Example 2: Conduct reads assembly, gene predition and annotation.

      $pgcgap --Assemble --ReadsPath <PATH> --reads1 .R1.clean.fastq.gz --reads2 .R2.clean.fastq.gz --kmmer 81 --genus bacillus --species thuringiensis --codon 11 --threads 4

    • Example 3: Constructing single core protein tree and core SNPs tree.

      $pgcgap --CoreTree --CDsPath <PATH> --AAsPath <PATH> --codon 11 --strain_num 3 --threads 4

    • Example 4: Conduct pan-genome analysis.

      $pgcgap --Pan --codon 11 --threads 4 --GffPath <PATH>

    • Example 5: Inference of orthologous gene groups.

      $pgcgap --orthoF --threads 4 --AAsPath <PATH>

    • Example 6: Compute whole-genome Average Nucleotide Identity (ANI).

      $pgcgap --ANI --threads 4 --queryL <FILE> --refL <FILE> --ANIO <FILE> --Scaf_suffix <STRING>

    • Example 7: Run COG annotation for each strain.

      $pgcgap --COG --threads 4 --AAsPath <PATH>

    • Example 8: Varients calling and phylogenetic tree construction based on reference genome.

      $pgcgap --VAR --threads 4 --refgbk <FILE> --ReadsPath <PATH> --reads1 .R1.clean.fastq.gz --reads2 .R2.clean.fastq.gz --suffix_len <INT> --strain_num <INT> --qualtype <STRING>

Generating Input files

Working directory

The directory where the PGCGAP software runs.

Assemble

Pair-end reads of all strains in a directory (default: ./Reads/ under the working directory).

ANI

QUERY_LIST and REFERENCE_LIST files containing full paths to genomes, one per line (default: scaf.list under the working directory). If the "–Assemble" fuction was run first, the list file will be generated automatically.

CoreTree

Amino acids file (With ".faa" as the suffix) and nucleotide (With ".ffn" as the suffix) file of each strain placed into two directorys (default: ./Results/Annotations/AAs/ and ./Results/Annotations/CDs/). The ".faa" and ".ffn" files of same strain should have the same prefix name. The name of protein IDs and gene IDs shoud be started with the strain name. The "Prokka" software was suggested to generate the input files. If the "–Assemble" fuction was run first, the files will be generated automatically.

OrthoF

A set of protein sequence files (one per species) in FASTA format under a directory (default: ./Results/Annotations/AAs/). If the "–Assemble" fuction was run first, the files will be generated automatically.

Pan

GFF3 files (With ".gff" as the suffix) of each strain placed into a directory. They must contain the nucleotide sequence at the end of the file. All GFF3 files created by Prokka are valid (default: ./Results/Annotations/GFF/). If the "–Assemble" fuction was run first, the files will be generated automatically.

COG

Amino acids file (With ".faa" as the suffix) of each strain placed into a directory (default: ./Results/Annotations/AAs/). If the "–Assemble" fuction was run first, the files will be generated automatically.

VAR

  • Pair-end reads of all strains in a directory (default: ./Reads/Over/ under the working directory).

  • The full path of reference genome in fasta format or genebank format (must be provided).

Output Files

Assemble

  • Results/Assembles/*_assembly
    Directorys contain assembly files and information of each strain.

  • Results/Assembles/Scaf
    Directory contain contig/scaffold of all strains.

  • Results/Annotations/*_annotation
    Directorys contain annotation files of each strain.

  • Results/Annotations/AAs
    Directory contain amino acids sequences of all strains.

  • Results/Annotations/CDs
    Directory contain nucleotide sequences of all strains.

  • Results/Annotations/GFF
    Directory contain the master annotation of all strains in GFF3 format.

ANI

  • Results/ANI/ANIs
    query genome, reference genome, ANI value, count of bidirectional fragment mappings, total query fragments

  • Results/ANI/ANIs.matrix
    file with identity values arranged in a phylip-formatted lower triangular matrix

  • Results/ANI/ANIs.heatmap
    An ANI matrix of all strains

  • Results/ANI/ANI_matrix.pdf
    The heatmap plot of "ANIs.heatmap"

CoreTree

  • Results/CoreTrees/faa/ALL.core.protein.fast
    Concatenated and aligned sequences file of single-core proteins.

  • Results/CoreTrees/faa2ffn/ALL.core.nucl.fasta
    Concatenated and aligned sequences file of single-core genes.

  • Results/CoreTrees/faa2ffn/ALL.core.snp.fasta
    Core SNPs of single-core genes in fasta format.

  • Results/CoreTrees/ALL.core.protein.nwk
    The phylogenetic tree file of single-copy proteins for all strains.

  • Results/CoreTrees/faa2ffn/ALL.core.snp.nwk
    The phylogenetic tree file of SNPs of single-copy core genes for all strains.

  • Results/CoreTrees/"Other_files"
    Intermediate directorys and files.

OrthoF

  • Results/OrthoFinder/Results_orthoF
    Same as OrthoFinder outputs

Pan

  • Results/PanGenome/Pangenome_Pie.pdf
    An 3D pie chart of the breakdown of genes and the number of isolate they are present in

  • Results/PanGenome/pangenome_frequency.pdf
    A graph with the frequency of genes versus the number of genomes

  • Results/PanGenome/Pangenome_matrix.pdf
    A figure showing the tree compared to a matrix with the presence and absence of core and accessory genes

  • Results/PanGenome/Other_files
    see roary outputs

COG

  • *.COG.xml, *.2gi.table, *.2id.table, *.2Sid.table
    Intermediate files

  • *.2Scog.table
    The super COG table of each strain

  • *.2Scog.table.pdf
    A plot of super COG table in pdf format

  • All_flags_relative_abundances.table A table containing the relative abundance of each flag for all strains

VAR

  • Results/Varients/directory-named-in-strains
    Directorys containing substitutions (snps) and insertions/deletions (indels) of each strain. See Snippy outputs for detail.

  • Results/Varients/Core
    The directory containing Core SNP phylogeny files

    • .aln : A core SNP alignment includes only SNP sites
    • .full.aln : A whole genome SNP alignment (includes invariant sites)
    • .nwk : Phylogenetic tree constructed with FastTree (ignoring possible recombination)
    • _tree.tre : Phylogenetic tree constructed with gubbins (get rid of recombination)

License

PGCGAP is free software, licensed under GPLv3.

Feedback and Issues

Please report any issues to the issues page or email us at liaochenlanruo@webmail.hzau.edu.cn.

Citation

If you use this software please cite: (Please keep an eye on it as it will be noted soon!)

FAQ

Q1 VAR founction ran failed to get annotated VCFs and Core results

Check the log file named in "strain_name.log" under Results/Varients// directory. If you find a sentence like "WARNING: All frames are zero! This seems rather odd, please check that 'frame' information in your 'genes' file is accurate." This is an snpEff error. Users can install JDK8 to solve this problem.


$conda install java-jdk=8.0.112

Click here for more solutions.

Q2 Could not determine version of minced please install version 2 or higher

When running prokka of Assemble founction, this error could happened, the error message shows as following:

Error: A JNI error has occurred, please check your installation and try again
Exception in thread "main" java.lang.UnsupportedClassVersionError: minced has been compiled by a more recent version of the Java Runtime (class file version 55.0), this version of the Java Runtime only recognizes class file versions up to 52.0
	at java.lang.ClassLoader.defineClass1(Native Method)
	at java.lang.ClassLoader.defineClass(ClassLoader.java:763)
	at java.security.SecureClassLoader.defineClass(SecureClassLoader.java:142)
	at java.net.URLClassLoader.defineClass(URLClassLoader.java:468)
	at java.net.URLClassLoader.access$100(URLClassLoader.java:74)
	at java.net.URLClassLoader$1.run(URLClassLoader.java:369)
	at java.net.URLClassLoader$1.run(URLClassLoader.java:363)
	at java.security.AccessController.doPrivileged(Native Method)
	at java.net.URLClassLoader.findClass(URLClassLoader.java:362)
	at java.lang.ClassLoader.loadClass(ClassLoader.java:424)
	at sun.misc.Launcher$AppClassLoader.loadClass(Launcher.java:349)
	at java.lang.ClassLoader.loadClass(ClassLoader.java:357)
	at sun.launcher.LauncherHelper.checkAndLoadMain(LauncherHelper.java:495)
[01:09:40] Could not determine version of minced - please install version 2.0 or higher

Users can downgrade the minced to version 0.3 to solve this problem.

$conda install minced=0.3

Click here for detail informations.

Q3 dyld: Library not loaded: @rpath/libcrypto.1.0.0.dylib

This error may happen when running function "VAR" on macOS. It is an error of openssl. Users can solve this problem as following:

#Firstly, install brew if have not installed before
$ruby -e "$(curl -fsSL https://raw.githubusercontent.com/Homebrew/install/master/install)"

#Install openssl with brew
brew install openssl

#Create the soft link for libraries
ln -s /usr/local/opt/openssl/lib/libcrypto.1.0.0.dylib /usr/local/lib/

ln -s /usr/local/opt/openssl/lib/libssl.1.0.0.dylib /usr/local/lib/

Click here for more informations

Q4 Use of uninitialized value in require at Encode.pm line 61

This warning may happen when running function "Pan". It is an warning of Roary software. The content of line 61 is "require Encode::ConfigLocal;". Users can ignore the warning. Click here for details:

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