DOC for V1.0.8

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Contents

Introduction

PGCGAP is a pipeline for prokaryotic comparative genomics analysis. It can take the pair-end reads, Oxford reads or PacBio 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 Windows WSL, Linux x64 platform and macOS. Because this software relies on a large number of other softwares, so it is recommended to install with Bioconda. Because PGCGAP relies on both Gubbins and Orthofinder, which are developed in different versions of python, Gubbins must be installed separately. Once Orthofinder was upgraded to python 3, PGCGAP can be installed with only one command.

Step1: Install Gubbins

If the system is installed with python 3 version of miniconda, Gubbins can be installed directly via conda.

$conda install gubbins

If the python 2 version of miniconda is installed on the system, users need to create a new python 3 environment to install Gubbins. And the Gubbins installation directory need to be added to the environment variable.

#Create an environment called gubbins

$conda create -n gubbins

#Activate the gubbins environment

$conda activate gubbins

#Installation of Gubbins

$conda install gubbins

View the installation path of gubbins and then add this path to the environment variable.

$whereis gubbins

Exit the gubbins environment

$conda deactivate

Step2: Install PGCGAP

$conda create -n pgcgap
$conda activate pgcgap
$conda install pgcgap
$conda deactivate

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

$conda activate pgcgap
$pgcgap --setup-COGdb
$conda deactivate

Required dependencies

Usage

  • Print the help messages: 
      $pgcgap --help


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


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


  • Functions:

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

    • [–Assemble] Assemble reads into contigs

    • [–Annotate] Genome annotation

    • [–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 )

    • [–MASH] Genome and metagenome similarity estimation using MinHash

    • [–pCOG] 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" "–VAR" and "–COG"] The total number of strains used for analysis, not including the reference genome

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

    • [–scafPath (PATH)] [Required by "–All", "–Annotate" and "–MASH"] Path for contigs/scaffolds (Default "Results/Assembles/Scaf/Illumina")

    • [–AAsPath (PATH)] [Required by "–All", "–CoreTree", "–OrthoF" and "–pCOG"] 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" )

    • [–Scaf_suffix (STRING)] [Required by "–All", "–Annotate" "MASH" and "–ANI"] The suffix of scaffolds or genomes. Here, "-8.fa" for Illumina data, ".contigs.fasta" for PacBio data and Oxford data. Users can also fill in other suffixes according to the actual situation (Default -8.fa)

    • [–codon (INT)] [Required by "–All", "–Annotate", "–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

      • [–platform (STRING)] Sequencing Platform, "illumina", "pacbio" and "oxford" available (Default illumina)

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

      • [–genomeSize (FLOAT)] [Required] An estimate of the size of the genome. Common suffices are allowed, for example, 3.7m or 2.8g. Needed by PacBio data and Oxford data (Default Unset)

    • –Annotate

      • [–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" ), if set to "NO", the SNPs of single-copy core genes will not be called

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

      • [-n (INT)] Word_length, -n 2 for thresholds 0.4-0.5, -n 3 for thresholds 0.5-0.6, -n 4 for thresholds 0.6-0.7, -n 5 for thresholds 0.7-1.0 ( 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 0.5 )

      • [-aS (FLOAT)] Alignment coverage for the shorter sequence. If set to 0.9, the alignment must covers 90% of the sequence ( Default 0.7 )

      • [-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" )


    • –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;

    • [–canu-bin (PATH)] Path to canu binary file. Default tries if canu 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.

    • [–mash-bin (PATH)] Path to the mash binary file. Default tries if mash 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 Escherichia coli as an example, total 6 strains for analysis.

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

      $pgcgap --All --platform illumina --ReadsPath Reads/Illumina --reads1 _1.fastq.gz --reads2 _2.fastq.gz --suffix_len 11 --kmmer 81 --genus Escherichia --species "Escherichia coli" --codon 11 --strain_num 6 --threads 4 --VAR --refgbk /mnt/h/PGCGAP_Examples/Reads/MG1655.gbff --qualtype sanger

    • Example 2: Genome assembly.

      • Illumina reads assembly

        In this dataset, the naming format of the genome is "strain_1.fastq.gz" and "strain_2.fastq.gz". The string after the strain name is "_1.fastq.gz", and its length is 11, so "–suffix_len" is set to 11.

       pgcgap --Assemble --platform illumina --ReadsPath Reads/Illumina --reads1 _1.fastq.gz --reads2 _2.fastq.gz --kmmer 81 --threads 4 --suffix_len 11

      • Oxford reads assembly

        Oxford nanopore only produces one reads file, so only the parameter of "–reads1" needs to be set, where the value is ".fasta". "–genomeSize" is the estimated genome size, and users can check the genome size of similar strains in NCBI database for reference. The parameter was set to "4.8m" here. The suffix of the reads file here is ".fasta" and its length is 6, so "–suffix_len" is set to 6.

       $pgcgap --Assemble --platform oxford --ReadsPath Reads/Oxford --reads1 .fasta --genomeSize 4.8m --threads 4 --suffix_len 6
      • PacBio reads assembly

      PacBio also produces only one reads file "pacbio.fastq", the parameter settings are similar to Oxford. The strain name is "pacbio" with the suffix ".fastq" and the suffix length is 6, so "–suffix_len" is set to 6.

       $pgcgap --Assemble --platform pacbio --ReadsPath Reads/PacBio --reads1 .fastq --genomeSize 4.8m --threads 4 --suffix_len 6

    • Example 3: Constructing single-copy core protein tree and core SNPs tree

       $pgcgap --CoreTree --CDsPath Results/Annotations/CDs --AAsPath Results/Annotations/AAs --codon 11 --strain_num 6 --threads 4

    • Example 4: Constructing single-copy core protein tree only.

      $pgcgap --CoreTree --CDsPath NO --AAsPath Results/Annotations/AAs --codon 11 --strain_num 6 --threads 4

    • Example 5: Conduct pan-genome analysis.

      $pgcgap --Pan --codon 11 --threads 4 --GffPath Results/Annotations/GFF

    • Example 6: Inference of orthologous gene groups.

      $pgcgap --OrthoF --threads 4 --AAsPath Results/Annotations/AAs

    • Example 7: Compute whole-genome Average Nucleotide Identity (ANI).

      $pgcgap --ANI --threads 4 --queryL scaf.list --refL scaf.list --ANIO Results/ANI/ANIs --Scaf_suffix .fa

    • Example 8: Genome and metagenome similarity estimation using MinHash

      $pgcgap --MASH --scafPath <PATH> --Scaf_suffix <STRING>

    • Example 9: Run COG annotation for each strain.

      $pgcgap --pCOG --threads 4 --strain_num 6 --AAsPath Results/Annotations/AAs

    • Example 10: Variants calling and phylogenetic tree construction based on reference genome.

      $pgcgap --VAR --threads 4 --refgbk /mnt/h/PGCGAP_Examples/Reads/MG1655.gbff --ReadsPath Reads/Illumina --reads1 _1.fastq.gz --reads2 _2.fastq.gz --suffix_len 11 --strain_num 6 --qualtype sanger

Generating Input files

Working directory

The directory where the PGCGAP software runs.

Assemble

Pair-end reads of all strains in a directory or PacBio reads or Oxford nanopore reads (Default: ./Reads/Illumina/ under the working directory).

Annotate

Genomes files (complete or draft) in a directory (Default: Results/Assembles/Scaf/Illumina 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" function 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 directories (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 should be started with the strain name. The "Prokka" software was suggested to generate the input files. If the "–Assemble" function was run first, the files will be generated automatically. If the "–CDsPath" was set to "NO", the nucleotide files will not be needed.

OrthoF

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

pCOG

Amino acids file (With ".faa" as the suffix) of each strain placed into a directory (default: ./Results/Annotations/AAs/). If the "–Assemble" function 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 GenBank format (must be provided).

Output Files

Assemble

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

  • Results/Assembles/PacBio
    Directories contain PacBio assembly files and information of each strain.

  • Results/Assembles/Illumina/Oxford
    Directories contain Oxford nanopore assembly files and information of each strain.

  • Results/Assembles/Scaf/Illumina
    Directory contains Illumina contigs/scaffolds of all strains.

  • Results/Assembles/Scaf/Oxford
    Directory contains Oxford nanopore contigs/scaffolds of all strains.

  • Results/Assembles/Scaf/PacBio
    Directory contains PacBio contigs/scaffolds of all strains.

Annotate

  • Results/Annotations/*_annotation
    directories 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
    The file contains comparation information of genome pairs. The document is composed of five columns, each of which represents 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.fasta
    Concatenated and aligned sequences file of single-copy core proteins.

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

  • Results/CoreTrees/faa2ffn/ALL.core.snp.fasta
    Core SNPs of single-copy 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 directories and files.

OrthoF

  • Results/OrthoFinder/Results_orthoF
    Same as OrthoFinder outputs

Pan

  • Results/PanGenome/Pangenome_Pie.pdf
    A 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

pCOG

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

  • Results/Variants/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/Variants// 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 a warning of Roary software. The content of line 61 is "require Encode::ConfigLocal;". Users can ignore the warning. Click here for details.

Updates

  • V1.0.3

    • Updated ANI fuction

  • V1.0.4

    • Add parallel for function "COG"
    • Optimized drawing of ANI heat map

  • V1.0.5

    • Bug repair for input of gubbins

  • V1.0.6

    • Modified CoreTree to split protein and SNPs tree constructing

  • V1.0.7

    • Split Assemble and Annotate into two functions
    • Added third generation genome assembly function
    • Changed the default parameters of the CoreTree function （aS 0.8 to 0.7 and aL 0.8 to 0.5）
    • Changed the name of function "COG" to "pCOG"
    • Fixed the sorting bug for ANI heat map

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